Non-cytotoxic protein conjugates

ABSTRACT

The present invention is directed to non-cytotoxic protein conjugates for inhibition or reduction of exocytic fusion in a nociceptive sensory afferent cell. The protein conjugates comprise: (i) a Targeting Moiety (TM), wherein the TM is an agonist of a receptor present on a nociceptive sensory afferent cell, and wherein the receptor undergoes endocytosis to be incorporated into an endosome within the nociceptive sensory afferent cell; (ii) a non-cytotoxic protease or a fragment thereof, wherein the protease or protease fragment is capable of cleaving a protein of the exocytic fusion to apparatus of the nociceptive sensory afferent cell; and (iii) a Translocation Domain, wherein the Translocation Domain translocates the protease or protease fragment from within the endosome, across the endosomal membrane, and into the cytosol of the nociceptive sensory afferent cell wherein the Targeting Moiety is selected from the group consisting of BAM, β-endorphin, bradykinin, substance P, dynorphin and/or nociceptin.

NAMES OF PARTIES TO A JOINT RESEARCH AGREEMENT

Allergan, Inc., a Delaware Corporation, and Syntaxin, Ltd., a UnitedKingdom corporation, are parties to a Joint Research Agreement.

FIELD OF THE INVENTION

This invention relates to a non-cytotoxic protein conjugate, and to theuse of said conjugate for treating pain.

BACKGROUND OF THE INVENTION

Toxins may be generally divided into two groups according to the type ofeffect that they have on a target cell. In more detail, the first groupof toxins kill their natural target cells, and are therefore known ascytotoxic toxin molecules. This group of toxins is is exemplified interalia by plant toxins such as ricin, and abrin, and by bacterial toxinssuch as diphtheria toxin, and Pseudomonas exotoxin A. Cytotoxic toxinstypically kill their target cells by inhibiting the cellular process ofprotein synthesis.

In contrast, the second group of toxins, which are known asnon-cytotoxic toxins, do not (as their name confirms) kill their naturaltarget cells. Non-cytotoxic toxins have attracted much less commercialinterest than have their cytotoxic counterparts, and exert their effectson a target cell by inhibiting cellular processes other than proteinsynthesis. As with their cytotoxic counterparts, non-cytotoxic toxinsare produced from a variety of sources such as plants, and bacteria.Bacterial non-cytotoxic toxins are now described in more detail.

Clostridial neurotoxins are proteins that typically have a molecularmass of the order of 150 kDa. They are produced by various species ofbacteria, especially of the genus Clostridium, most importantly C.tetani and several strains of C. botulinum, C. butyricum and C.argentinense. There are at present eight different classes of theclostridial neurotoxin, namely: tetanus toxin, and botulinum neurotoxinin its serotypes A, B, C₁, D, E, F and G, and they all share similarstructures and modes of action.

Clostridial neurotoxins represent a major group of non-cytotoxic toxinmolecules, and are synthesised by the host bacterium as singlepolypeptides that are modified post-translationally by a proteolyticcleavage event to form two polypeptide chains joined together by adisulphide bond. The two chains are termed the heavy chain (H-chain),which has a molecular mass of approximately 100 kDa, and the light chain(L-chain), which has a molecular mass of approximately 50 kDa.

L-chains possess a protease function (zinc-dependent endopeptidaseactivity) and exhibit high substrate specificity for vesicle and/orplasma membrane associated proteins involved in the exocytic process.L-chains from different clostridial species or serotypes may hydrolysedifferent but specific peptide bonds in one of three substrate proteins,namely synaptobrevin, syntaxin or SNAP-25. These substrates areimportant components of the neurosecretory machinery.

Non-cytotoxic toxins are also produced by other bacteria, such as fromthe genus Neisseria, most importantly from the species N. gonorrhoeae.For example, Neisseria sp. produces the non-cytotoxic toxin IgA protease(see WO99/58571).

It has been well documented in the art that toxin molecules may bere-targeted to a cell that is not the toxin's natural target cell. Whenso re-targeted, the modified toxin is capable of binding to a desiredtarget cell and, following subsequent translocation into the cytosol, iscapable of exerting its effect on the target cell. Said re-targeting isachieved by replacing the natural Targeting Moiety (TM) of the toxinwith a different TM. In this regard, the TM is selected so that it willbind to a desired target cell, and allow subsequent passage of themodified toxin into an endosome within the target cell. The modifiedtoxin also comprises a translocation domain to enable entry of thenon-cytotoxic protease into the cell cytosol. The translocation domaincan be the natural translocation domain of the toxin or it can be adifferent translocation domain obtained from a microbial protein withtranslocation activity.

For example, in the context of non-cytotoxic toxin molecules, it hasbeen well documented that a clostridial neurotoxin may be re-targeted byincorporation of a Targeting Moiety (TM), which is not the natural TM ofa clostridial neurotoxin. The described chemical conjugation andrecombinant methodologies are now regarded as conventional, andreference is made to Hermanson, G. T. (1996), Bioconjugate techniques,Academic Press, and to Wong, S. S. (1991), Chemistry of proteinconjugation and cross-linking, CRC Press.

For example, WO94/21300 describes modified clostridial neurotoxinmolecules that are capable of regulating Integral Membrane Protein (IMP)density present at the cell surface of the target cell. The modifiedneurotoxin molecules are thus capable of controlling cell activity (e.g.glucose uptake) of the target cell. WO96/33273 and WO99/17806 describemodified clostridial neurotoxin molecules that target peripheral sensoryafferents. The modified neurotoxin molecules are thus capable ofdemonstrating an analgesic effect. WO00/10598 describes the preparationof modified clostridial neurotoxin molecules that target mucushypersecreting cells (or neuronal cells controlling said mucushypersecreting cells), which modified neurotoxins are capable ofinhibiting hypersecretion from said cells. WO01/21213 describes modifiedclostridial neurotoxin molecules that target a wide range of differenttypes of non-neuronal target cells. The modified molecules are thuscapable of preventing secretion from the target cells. Additionalpublications in the technical field of re-targeted toxin moleculesinclude: WO00/62814; WO00/04926; U.S. Pat. No. 5,773,586; WO93/15766;WO00/61192; and WO99/58571.

Thus, from the above-described publications, it will be appreciated thatthe basic concept of re-targeting a non-cytotoxic protease to a desiredtarget cell, by selecting a TM that has a corresponding receptor presenton the target cell, has been well documented.

However, different receptors present on a target cell of interestdemonstrate different binding affinities for different TMs. This may bea particular problem with pain-sensing cells, which possess a wide rangeof receptor types having different binding affinities for different TMs.Thus, a re-targeted conjugate comprising a particular TM (that binds toa receptor on a pain-sensing cell) may demonstrate a low bindingaffinity for a pain-sensing target cell, which is undesirable.

There is therefore a need to develop modified non-cytotoxic conjugatesthat address one or more of the above problems. Of particular interestis the development of an improved conjugate for use in treating pain.

SUMMARY OF THE INVENTION

The present invention seeks to address one or more of the above problemsby providing unique non-cytotoxic protein conjugates. In one embodiment,the Targeting Moiety (TM) component employed with a non-cytotoxicprotein conjugate of the present invention is an “agonist” of a receptorthat is present on the pain-sensing target cell of interest. In oneembodiment, the pain-sensing target cell is a nociceptive sensoryafferent, for example a primary nociceptive sensory afferent.

Accordingly, in a first aspect, the present invention provides anon-cytotoxic conjugate for inhibition or reduction of exocytic fusionin a nociceptive sensory afferent cell, is comprising:

-   -   (i) a Targeting Moiety (TM), wherein said TM is capable of        binding to a Binding site on a nociceptive sensory afferent        cell, and wherein said Binding site undergoes endocytosis to be        incorporated into an endosome within the nociceptive sensory        afferent cell;    -   (ii) a non-cytotoxic protease or a fragment thereof, wherein the        protease or protease fragment is capable of cleaving a protein        of the exocytic fusion apparatus of said nociceptive sensory        afferent cell; and    -   (iii) a Translocation Domain, wherein the Translocation Domain        translocates the protease or protease fragment from within the        endosome, across the endosomal membrane, and into the cytosol of        the nociceptive sensory afferent cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—Expression and Purification of recLH_(N)/B Fusion Protein

SDS-PAGE analysis of expression and purification of recLH_(N)/B from E.coli. In FIG. 1, recLH_(N)/B is purified from cell paste using a threecolumn strategy as described in Example 3. Protein samples are separatedby SDS-PAGE and visualised by staining with simplyblue safestaincoomassie reagent. Crude, soluble MBP-LH_(N)/B fusion protein containedwithin the clarified extract (lane 2) is loaded onto Q-Sepharose FFanion-exchange resin. Lane 3 represents recombinant MBP-LH_(N)/B fusioneluted from column at 150-200 mM salt. This sample is treated withfactor Xa protease to remove MBP affinity tag (lane 4), and cleavedmixture diluted to lower salt concentration prior to loading onto aQ-Sepharose FF anion-exchange column. Material eluted between 120-170 mMsalt was rich in LH_(N)/B (lane 5). Protein in lanes 6 and 8 representsLH_(N)/B harvested after treatment with enterokinase and finalpurification using Benzamidine Sepharose, under non-reducing andreducing conditions respectively. Lanes 1 and 7 represent molecular massmarkers [Mark 12 (Invitrogen)].

FIG. 2—Expression and Purification of LH_(N)/C Fusion Protein

SDS-PAGE analysis of expression and purification of LH_(N)/C from E.coli. In FIG. 2, recLH_(N)/C is purified from E. coli cell paste using atwo-step strategy described in Example 4. Protein samples are separatedby SDS-PAGE and visualised by staining with coomassie blue. ClarifiedCrude cell lysate (lane 2) is loaded onto Q-Sepharose FF anion-exchangeresin. Fusion protein, MBP-LH_(N)/C is eluted with 0.1 M NaCl (lane 3).Eluted material incubated at 22° C. for 16 h with factor Xa protease(New England Biolabs) to cleave fusion tag MBP and nick recLH_(N)/C atthe linker site. The protein of interest is further purified fromcleaved fusion products (lane 4) using Q-Sepharose FF. Lanes 5 and 7show purified recLH_(N)/C under non-reducing conditions and reduced with10 mM DTT respectively, to illustrate disulphide bonding at the linkerregion between LC and H_(N) domains after nicking with factor Xa. Lanes1 and 6 represent molecular mass markers (shown in KDa); Mark 12(Invitrogen).

FIG. 3—Expression and Purification of N[1-17]-LH_(N)/A Fusion Protein

SDS-PAGE analysis of expression and purification of N[1-17]-LH_(N)/Afrom E. coli. In FIG. 3, N[1-17]-LH_(N)/A is purified from E. coli BL21cell paste using the methodology outlined in Example 9. Briefly, thesoluble products obtained following cell disruption were applied to anickel-charged affinity capture column. Bound proteins were eluted with100 mM imidazole, treated with Factor Xa to activate the fusion proteinand remove the maltose-binding protein (MBP) tag, then re-applied to asecond nickel-charged affinity capture column. Samples from thepurification procedure were assessed by SDS-PAGE (Panel A) and Westernblotting (Panel B). Anti-nociceptin antisera (obtained from Abcam) wereused as the primary antibody for Western blotting. The final purifiedmaterial in the absence and presence of reducing agent is identified inthe lanes marked [−] and [+] respectively.

FIG. 4—Purification of a LC/A-Nociceptin-H_(N)/A Fusion Protein

Using the methodology outlined in Example 26, a LC/A-nociceptin-H_(N)/Afusion protein was purified from E. coli BL21 cells. Briefly, thesoluble products obtained following cell disruption were applied to anickel-charged affinity capture column. Bound proteins were eluted with100 mM imidazole, treated with Factor Xa to activate the fusion proteinand remove the maltose-binding protein (MBP) tag, then re-applied to asecond nickel-charged affinity capture column. Samples from thepurification procedure were assessed by SDS-PAGE (Panel A) and Westernblotting (Panel B). Anti-nociceptin is antisera (obtained from Abcam)were used as the primary antibody for Western blotting. The finalpurified material in the absence and presence of reducing agent isidentified in the lanes marked [−] and [+] respectively.

FIG. 5—Purification of a Nociceptin-LC/A-H_(N)/A Fusion Protein

Using the methodology outlined in Example 26, a nociceptin-LC/A-H_(N)/Afusion protein was purified from E. coli BL21 cells. Briefly, thesoluble products obtained following cell disruption were applied to anickel-charged affinity capture column. Bound proteins were eluted with100 mM imidazole, treated with Factor Xa to activate the fusion proteinand remove the maltose-binding protein (MBP) tag, then re-applied to asecond nickel-charged affinity capture column. Samples from thepurification procedure were assessed by SDS-PAGE (Panel A) and Westernblotting (Panel B). Anti-nociceptin antisera (obtained from Abcam) wereused as the primary antibody for Western blotting. The final purifiedmaterial in the absence and presence of reducing agent is identified inthe lanes marked [−] and [+] respectively.

FIG. 6—Purification of a LC/C-Nociceptin-H_(N)/C Fusion Protein

Using the methodology outlined in Example 26, an LC/C-nociceptin-H_(N)/Cfusion protein was purified from E. coli BL21 cells. Briefly, thesoluble products obtained following cell disruption were applied to anickel-charged affinity capture column. Bound proteins were eluted with100 mM imidazole, treated with Factor Xa to activate the fusion proteinand remove the maltose-binding protein (MBP) tag, then re-applied to asecond nickel-charged affinity capture column. Samples from thepurification procedure were assessed by SDS-PAGE (Panel A) and Westernblotting (Panel B). Anti-nociceptin antisera (obtained from Abcam) wereused as the primary antibody for Western blotting. The final purifiedmaterial in the absence and presence of reducing agent is identified inthe lanes marked [−] and [+] respectively.

FIG. 7—Purification of a LC/A-met Enkephalin-H_(N)/A Fusion Protein

Using the methodology outlined in Example 26, an LC/A-metenkephalin-H_(N)/A fusion protein was purified from E. coli BL21 cells.Briefly, the soluble products obtained following cell disruption wereapplied to a nickel-charged affinity capture column. Bound proteins wereeluted with 100 mM imidazole, treated with Factor Xa to activate thefusion protein and remove the maltose-binding protein (MBP) tag, thenre-applied to a second nickel-charged affinity capture column. Samplesfrom the purification procedure were assessed by SDS-PAGE. The finalpurified material in the absence and presence of reducing agent isidentified in the lanes marked [−] and [+] respectively.

FIG. 8—Comparison of Binding Efficacy of a LC/A-Nociceptin-H_(N)/AFusion Protein and a Nociceptin-LC/A-H_(N)/A Fusion Protein

The ability of nociceptin fusions to bind to the ORL₁ receptor wasassessed using a simple competition-based assay. Primary cultures ofdorsal root ganglia (DRG) were exposed to varying concentrations of testmaterial in the presence of 1 nM [3H]-nociceptin. The reduction inspecific binding of the radiolabelled ligand was assessed byscintillation counting, and plotted in comparison to the efficacy ofunlabelled ligand (Tocris nociceptin). It is clear that theLC/A-nociceptin-H_(N)/A fusion is far superior to thenociceptin-LC/A-H_(N)/A fusion at interacting with the ORL₁ receptor.

FIG. 9—In vitro catalytic activity of a LC/A-nociceptin-H_(N)/A fusionprotein

The in vitro endopeptidase activity of the purifiedLC/A-nociceptin-H_(N)/A fusion protein was determined essentially asdescribed in Chaddock et al 2002, Prot. Express Purif. 25, 219-228.Briefly, SNAP-25 peptide immobilised to an ELISA plate was exposed tovarying concentrations of fusion protein for 1 hour at 37° C. Followinga series of washes, the amount of cleaved SNAP-25 peptide was quantifiedby reactivity with a specific antisera.

FIG. 10—Purification of a LC/A-Nociceptin Variant-H_(N)/A Fusion Protein

Using the methodology outlined in Example 26, an LC/A-nociceptinvariant-H_(N)/A fusion protein was purified from E. coli BL21 cells.Briefly, the soluble products obtained following cell disruption wereapplied to a nickel-charged affinity capture column. Bound proteins wereeluted with 100 mM imidazole, treated with Factor Xa to activate thefusion protein and remove the maltose-binding protein (MBP) tag, thenre-applied to a second nickel-charged affinity capture column. Samplesfrom the purification procedure were assessed by SDS-PAGE. The finalpurified material in the absence and presence of reducing agent isidentified in the lanes marked [−] and [+] respectively.

FIG. 11—Comparison of Binding Efficacy of a LC/A-Nociceptin-H_(N)/AFusion Protein and a LC/A-Nociceptin Variant-H_(N)/A Fusion Protein

The ability of nociceptin fusions to bind to the ORL₁ receptor wasassessed using a simple competition-based assay. Primary cultures ofdorsal root ganglia (DRG) were exposed to varying concentrations of testmaterial in the presence of 1 nM [3H]-nociceptin. The reduction inspecific binding of the radiolabelled ligand was assessed byscintillation counting, and plotted in comparison to the efficacy ofunlabelled ligand (Tocris nociceptin). It is clear that theLC/A-nociceptin variant-H_(N)/A fusion (CPNv-LHA) is superior to theLC/A-nociceptin variant-H_(N)/A fusion (CPN-LHA) at interacting with theORL₁ receptor.

FIG. 12—Expressed/Purified LC/A-Nociceptin-H_(N)/A Fusion Protein Familywith Variable Spacer Length Product(s)

Using the methodology outlined in Example 26, variants of theLC/A-CPN-H_(N)/A fusion consisting of GS10, GS30 and HX27 are purifiedfrom E. coli cell paste. Samples from the purification ofLC/A-CPN(GS10)-H_(N)/A, LC/A-CPN(GS15)-H_(N)/A, LC/A-CPN(GS25)-H_(N)/A,LC/A-CPN(GS30)-H_(N)/A and LC/A-CPN(HX27)-H_(N)/A were assessed bySDS-PAGE prior to staining with Coomassie Blue. The electrophoresisprofile indicates purification of a disulphide-bonded di-chain speciesof the expected molecular mass of CPBE-A. Top panel: M=benchmarkmolecular mass markers; S=total E. coli protein soluble fraction;FT=proteins that did not bind to the Ni²⁺-charged Sepharose column;FUSION=fusion protein eluted by the addition of imidazole. Bottom panel:Lane 1=benchmark molecular mass markers; Lane 2=total E. coli proteinsoluble fraction; Lane 3=purified material following initial capture onNi²⁺-charged Sepharose; Lane 4=Factor Xa treated material prior to finalcapture on Ni²⁺-charged Sepharose; Lane 5=purified final material postactivation with Factor Xa (5 μl); Lane 6=purified final material postactivation with Factor Xa (10 μl); Lane 7=purified final material postactivation with Factor Xa (20 μl); Lane 8=purified final material postactivation with Factor Xa+DTT (5 μl); Lane 9=purified final materialpost activation with Factor Xa+DTT (10 μl); Lane 10=purified finalmaterial post activation with Factor Xa+DTT (20 μl).

FIG. 13—Inhibition of SP Release and Cleavage of SNAP-25 by CPN-A

Briefly, primary cultures of dorsal root ganglia (DRG) were exposed tovarying concentrations of CPN-A for 24 hours. Cellular proteins wereseparated by SDS-PAGE, Western blotted, and probed with anti-SNAP-25 tofacilitate an assessment of SNAP-25 cleavage. The percentage of cleavedSNAP-25 was calculated by densitometric analysis and plotted againstfusion concentration (dashed line). Material was also recovered for ananalysis of substance P content using a specific EIA kit. Inhibition ofsubstance P release is illustrated by the solid line. The fusionconcentration required to achieve 50% maximal SNAP-25 cleavage isestimated to be 6.30±2.48 nM.

FIG. 14—Inhibition of SP Release and Cleavage of SNAP-25 Over ExtendedTime Periods after Exposure of DRG to CPN-A

Primary cultures of dorsal root ganglia (DRG) were exposed to varyingconcentrations of CPN-A for 24 hours. Botulinum neurotoxin (BoNT/A) wasused as a control. After this initial exposure, extracellular materialwas removed by washing, and the cells incubated at 37° C. for varyingperiods of time. At specific time points, cellular proteins wereseparated by SDS-PAGE, Western blotted, and probed with anti-SNAP-25 tofacilitate an assessment of SNAP-25 cleavage. The percentage of cleavedSNAP-25 was calculated by densitometric analysis and illustrated by thedotted lines. Material was also recovered for an analysis of substance Pcontent using a specific EIA kit. Inhibition of substance P release isillustrated by the solid lines.

FIG. 15—Cleavage of SNAP-25 by CPNv-A

Primary cultures of dorsal root ganglia (DRG) were exposed to varyingconcentrations of CPNv-A for 24 hours. Cellular proteins were separatedby SDS-PAGE, Western blotted, and probed with anti-SNAP-25 to facilitatean assessment of SNAP-25 cleavage. The percentage of cleaved SNAP-25 wascalculated by densitometric analysis. The fusion concentration requiredto achieve 50% maximal SNAP-25 cleavage is estimated to be 1.38±0.36 nM.

FIG. 16—Cleavage of SNAP-25 Over Extended Time Periods after Exposure ofDRG to CPNv-A

Primary cultures of dorsal root ganglia (DRG) were exposed to varyingconcentrations of CPNv-A for 24 hours. CPN-A was used as a control.After this initial exposure, extracellular material was removed bywashing, and the cells incubated at 37° C. for varying periods of time.At specific time points, cellular proteins were separated by SDS-PAGE,Western blotted, and probed with anti-SNAP-25 to facilitate anassessment of SNAP-25 cleavage. The percentage of cleaved SNAP-25 wascalculated by densitometric analysis.

FIG. 17—CPNv-A Fusion-Mediated Displacement of [3H]-Nociceptin Binding

The ability of nociceptin fusions to bind to the ORL₁ receptor wasassessed using a simple competition-based assay. Primary cultures ofdorsal root ganglia (DRG) were exposed to varying concentrations of testmaterial in the presence of 1 nM [3H]-nociceptin. The reduction inspecific binding of the radiolabelled ligand was assessed byscintillation counting, and plotted in comparison to the efficacy ofunlabelled ligand (Tocris nociceptin). It is clear that theLC/A-nociceptin variant-H_(N)/A fusion (labelled as CPNv-LHnA) issuperior to the LC/A-nociceptin-H_(N)/A fusion (labelled as CPN-LHnA) atinteracting with the ORL₁ receptor.

FIG. 18-Expressed/Purified CPNv(Ek)-A Product

Proteins were subjected to SDS-PAGE prior to staining with CoomassieBlue. The electrophoresis profile indicates purification of adisulphide-bonded di-chain species of the expected molecular mass ofCPNv(Ek)-A. Lane 1=benchmark molecular mass markers; Lane 2=total E.coli protein soluble fraction; Lane 3=purified material followinginitial capture on Ni²⁺-charged Sepharose; Lane 4=purified finalmaterial post activation with enterokinase (5 μl); Lane 5=purified finalmaterial post activation with enterokinase (10 μl); Lane 6=purifiedfinal material post activation with enterokinase (20 μl); Lane7=purified final material post activation with enterokinase+DTT (5 μl);Lane 8=purified final material post activation with enterokinase+DTT (10μl); Lane 9=purified final material post activation withenterokinase+DTT (20 μl).

FIG. 19—Cleavage of SNAP-25 by CPNv(Ek)-A

Primary cultures of dorsal root ganglia (DRG) were exposed to varyingconcentrations of CPNv(Ek)-A for 24 hours. Cellular proteins wereseparated by SDS-PAGE, Western blotted, and probed with anti-SNAP-25 tofacilitate an assessment of SNAP-25 cleavage. The percentage of cleavedSNAP-25 was calculated by densitometric analysis. CPNv-A as prepared inExample 26 was used for comparison purposes. The percentage cleavage ofSNAP-25 by CPNv(Ek)-A (labelled as En activated) and CPNv-A (labelled asXa activated) are illustrated.

FIG. 20-Expressed/Purified CPNv-C Product

Proteins were subjected to SDS-PAGE prior to staining with CoomassieBlue. The electrophoresis profile indicates purification of adisulphide-bonded di-chain species of the expected molecular mass ofCPNv-C. Lane 1=benchmark molecular mass markers; Lane 2=total E. coliprotein soluble fraction; Lane 3=purified material following initialcapture on Ni²⁺-charged Sepharose; Lane 4=Factor Xa treated materialprior to final capture on Ni²⁺-charged Sepharose; Lane 5=purifiedmaterial following second capture on Ni²⁺-charged Sepharose; Lane6=final purified material; Lane 7=final purified material+DTT; Lane8=benchmark molecular mass markers.

FIG. 21—Cleavage of Syntaxin by CPNv-C

Primary cultures of dorsal root ganglia (DRG) were exposed to varyingconcentrations of CPNv-C for 24 hours. Cellular proteins were separatedby SDS-PAGE, Western blotted, and probed with anti-syntaxin tofacilitate an assessment of syntaxin cleavage. The percentage of cleavedsyntaxin was calculated by densitometric analysis. The fusionconcentration required to achieve 50% maximal syntaxin cleavage isestimated to be 3.13±1.96 nM.

FIG. 22—CPN-A Efficacy in the Acute Capsaicin-Induced MechanicalAllodynia Model

The ability of an LC/A-nociceptin-H_(N)/A fusion (CPN/A) to inhibitcapsaicin-induced mechanical allodynia was evaluated followingsubcutaneous intraplantar injection in the rat hind paw. Test animalswere evaluated for paw withdrawal frequency (PWF %) in response to a 10g Von Frey filament stimulus series (10 stimuli×3 trials) prior torecruitment into the study (Pre-Treat); after subcutaneous intraplantartreatment with CPN/A but before capsaicin (Pre-CAP); and followingcapsaicin challenge post-injection of CPN/A (average of responses at 15′and 30′; CAP). Capsaicin challenge was achieved by injection of 10 μL ofa 0.3% solution. Sample dilutions were prepared in 0.5% BSA/saline.

FIG. 23—CPN-A Efficacy in the Streptozotocin (STZ)-Induced PeripheralDiabetic Neuropathy (Neuropathic Pain) Model

Male Sprague-Dawley rats (250-300 g) are treated with 65 mg/kg STZ incitrate buffer (I.V.) and blood glucose and lipid are measured weekly todefine the readiness of the model. Paw Withdrawal Threshold (PWT) ismeasured in response to a Von Frey filament stimulus series over aperiod of time. Allodynia is said to be established when the PWT on twoconsecutive test dates (separated by 1 week) measures below 6 g on thescale. At this point, rats are randomized to either a saline group(negative efficacy control), gabapentin group (positive efficacycontrol) or a test group (CPN/A). Test materials (20-25 μl) are injectedsubcutaneously as a single injection (except gabapentin) and the PWT ismeasured at 1 day post-treatment and periodically thereafter over a 2week period. Gabapentin (30 mg/kg i.p. @3 ml/kg injection volume) isinjected daily, 2 hours prior to the start of PWT testing.

FIG. 24—CPNv-A Efficacy in the Acute Capsaicin-Induced MechanicalAllodynia Model

The ability of an LC/A-nociceptin variant-H_(N)/A fusion (CPNv/A) toinhibit capsaicin-induced mechanical allodynia was evaluated followingsubcutaneous intraplantar injection in the rat hind paw. Test animalswere evaluated for paw withdrawal frequency (PWF %) in response to a 10g Von Frey filament stimulus series (10 stimuli×3 trials) prior torecruitment into the study (Pre-Treat), after subcutaneous intraplantartreatment with CPNv/A but before capsaicin (Pre-CAP), and followingcapsaicin challenge post-injection of CPNv/A (average of responses at15′ and 30′; CAP). Capsaicin challenge was achieved by injection of 10μL of a 0.3% solution. Sample dilutions were prepared in 0.5%BSA/saline. These data are expressed as a normalized paw withdrawalfrequency differential, in which the difference between the peakresponse (post-capsaicin) and the baseline response (pre-capsaicin) isexpressed as a percentage. With this analysis, it can be seen thatCPNv/A is more potent than CPN/A since a lower dose of CPNv/A isrequired to achieve similar analgesic effect to that seen with CPN/A.

FIG. 25—Expressed/Purified LC/A-CPLE-H_(N)/A Product

Proteins were subjected to SDS-PAGE prior to staining with CoomassieBlue. The electrophoresis profile indicates purification of adisulphide-bonded di-chain species of the expected molecular mass ofCPLE-A. Lane 1=benchmark molecular mass markers; Lane 2=total E. coliprotein soluble fraction; Lane 3=purified material following initialcapture on Ni²⁺-charged Sepharose; Lane 4=Factor Xa treated materialprior to final capture on Ni²⁺-charged Sepharose; Lane 5=purifiedmaterial following second capture on Ni²⁺-charged Sepharose; Lane6=final purified material; Lane 7=final purified material+DTT.

FIG. 26—Expressed/Purified LC/A-CPBE-H_(N)/A Product

Proteins were subjected to SDS-PAGE prior to staining with CoomassieBlue. The electrophoresis profile indicates purification of adisulphide-bonded di-chain species of the expected molecular mass ofCPBE-A. Lane 1=total E. coli protein soluble fraction; Lane 2=purifiedmaterial following initial capture on Ni²⁺-charged Sepharose; Lane3=Factor Xa treated material prior to final capture on Ni²⁺-chargedSepharose; Lane 4=purified final material post activation with Factor Xa(5 μl); Lane 5=purified final material post activation with Factor Xa(10 μl); Lane 6=purified final material post activation with Factor Xa(20 μl); Lane 7=purified final material post activation with FactorXa+DTT (5 μl); Lane 8=purified final material post activation withFactor Xa+DTT (10 μl); Lane 9=purified final material post activationwith Factor Xa+DTT (20 μl); Lane 10=benchmark molecular mass markers.

FIG. 27—Expressed/Purified CPOP-A Product

Proteins were subjected to SDS-PAGE prior to staining with CoomassieBlue. The electrophoresis profile indicates purification of adisulphide-bonded di-chain species of the expected molecular mass ofCPOP-A. Lane 1=benchmark molecular mass markers; Lane 2=purifiedmaterial following initial capture on Ni²⁺-charged Sepharose; Lane3=Factor Xa treated material prior to final capture on Ni²⁺-chargedSepharose; Lane 4=purified material following second capture onNi²⁺-charged Sepharose; Lane 5=purified final material post activationwith Factor Xa (5 μl); Lane 6=purified final material post activationwith Factor Xa (10 μl); Lane 7=purified final material post activationwith Factor Xa (20 μl); Lane 8=purified final material post activationwith Factor Xa+DTT (5 μl); Lane 9=purified final material postactivation with Factor Xa+DTT (10 μl); Lane 10=purified final materialpost activation with Factor Xa+DTT (20 μl).

FIG. 28—Expressed/Purified CPOPv-A Product

Proteins were subjected to SDS-PAGE prior to staining with CoomassieBlue. The electrophoresis profile indicates purification of adisulphide-bonded di-chain species of the expected molecular mass ofCPOPv-A. Lane 1=benchmark molecular mass markers; Lane 2=total E. coliprotein soluble fraction; Lane 3=purified material following initialcapture on Ni²⁺-charged Sepharose; Lane 4=Factor Xa treated materialprior to final capture on Ni²⁺-charged Sepharose; Lane 5=purified finalmaterial post activation with Factor Xa (5 μl); Lane 6=purified finalmaterial post activation with Factor Xa (10 μl); Lane 7=purified finalmaterial post activation with Factor Xa (20 μl); Lane 8=purified finalmaterial post activation with Factor Xa+DTT (5 μl); Lane 9=purifiedfinal material post activation with Factor Xa+DTT (10 μl); Lane10=purified final material post activation with Factor Xa+DTT (20 μl).

FIG. 29—In Vitro SNAP-25 Cleavage in a DRG Cell Model

Primary cultures of dorsal root ganglia (DRG) were exposed to varyingconcentrations of CPOPv-A for 24 hours. Cellular proteins were separatedby SDS-PAGE, Western blotted, and probed with anti-SNAP-25 to facilitatean assessment of SNAP-25 cleavage. The percentage of cleaved SNAP-25 wascalculated by densitometric analysis.

FIG. 30—Expressed/Purified CPNv-A-FXa-HT (Removable His-Tag)

Proteins were subjected to SDS-PAGE prior to staining with CoomassieBlue. The electrophoresis profile indicates purification of adisulphide-bonded di-chain species of the expected molecular mass ofCPNv-A-FXa-HT. Lane 1=benchmark molecular mass markers; Lane 2=total E.coli protein soluble fraction; Lane 3=Factor Xa treated material priorto final capture on Ni²⁺-charged Sepharose; Lane 4=purified finalmaterial post activation with Factor Xa; Lane 5=purified final materialpost activation with Factor Xa+DTT.

FIG. 31—In Vitro Efficacy of LC/A-Nociceptin-H_(N)/A Fusion Proteinswith Variable Spacer Length, as Assessed by Ligand Competition Assay

The ability of LC/A-nociceptin-H_(N)/A fusions of variable spacer lengthto bind to the ORL₁ receptor was assessed using a simplecompetition-based assay. Primary cultures of dorsal root ganglia (DRG)were exposed to varying concentrations of test material in the presenceof 1 nM [3H]-nociceptin. The reduction in specific binding of theradiolabelled ligand was assessed by scintillation counting, and plottedin comparison to the efficacy of unlabelled ligand (Tocris nociceptin).The upper panel illustrates the displacement characteristics of the GS0,GS20, GS30 and Hx27 spacers, whilst the lower panel illustrates thedisplacement achieved by the GS10, GS15 and GS25 spaced fusion proteins.It is concluded that the GS0 and GS30 spacers are ineffective, and theGS10 is poorly effective, at displacing nociceptin from the ORL1receptor.

FIG. 32—In Vitro Efficacy of LC/A-Nociceptin-H_(N)/A Fusion Proteinswith Variable Spacer Length, as Assessed by In Vitro SNAP-25 Cleavage

Primary cultures of dorsal root ganglia (DRG) were exposed to varyingconcentrations of CPN-A (of variable spacer length) for 24 hours.Cellular proteins were separated by SDS-PAGE, Western blotted, andprobed with anti-SNAP-25 to facilitate an assessment of SNAP-25cleavage. The percentage of cleaved SNAP-25 was calculated bydensitometric analysis. The poorly effective binding characteristics ofthe GS10 spaced fusion protein (see FIG. 28) are reflected in the higherconcentrations of fusion required to achieve cleavage of intracellularSNAP-25. GS0 and GS30 spaced fusion proteins were completely ineffective(date not shown). GS15, 20 and 25 spaced fusion proteins were similarlyeffective.

FIG. 33—Cleavage of SNARE Protein by Dynorphin Conjugates in EmbryonicSpinal Cord Neurons (eSCNs)

Embryonic spinal cord neurons were exposed to varying concentrations ofdynorphin conjugates of the present invention for 24 hours. Cellularproteins were separated by SDS-PAGE, Western blotted, and probed withanti-SNAP-25 to facilitate an assessment of SNAP-25 cleavage. Thepercentage of cleaved SNAP-25 was calculated by densitometric analysis.It is clear that LC/A-dynorphin-H_(N)/A fusion is more potent than anunliganded LC/A-H_(N)/A control molecule. The concentration ofLC/A-dynorphin-H_(N)/A fusion required to achieve 50% maximal SNAP-25cleavage is estimated to be 35.3 nM and the concentration for theLC/A-H_(N)/A control required to achieve 50% maximal SNAP-25 cleavagecould not be determined due to it's low potency.

FIG. 34—Cleavage of SNARE Protein by Dynorphin Conjugates in ChineseHamster Ovary Cells (CHO-K1 Cells) Transfected with OP2 Receptor andSNAP-25

Chinese hamster ovary (CHO) cells were transfected so that they expressthe OP2 receptor. Said cells were further transfected to express a SNAREprotein (SNAP-25). The transfected cells were exposed to varyingconcentrations of different dynorphin conjugates for 24 hours. Cellularproteins were separated by SDS-PAGE, Western blotted, and probed withanti-SNAP-25 to facilitate an assessment of SNAP-25 cleavage. Thepercentage of cleaved SNAP-25 was calculated by densitometric analysis.It is clear that LC/A-CPDY-H_(N)/A conjugates are more potent than theunliganded LC/A-H_(N)/A control molecule (labelled as LC/A-H_(N)/A).

FIG. 35—Cleavage of SNARE Protein by Dynorphin Conjugates in EmbryonicSpinal Cord Neurons (eSCNs)

Embryonic spinal cord neurons were exposed to varying concentrations ofdynorphin conjugates of the present invention for 24 hours. Cellularproteins were separated by SDS-PAGE, Western blotted, and probed withanti-SNAP-25 to facilitate an assessment of SNAP-25 cleavage. Thepercentage of cleaved SNAP-25 was calculated by densitometric analysis.It is clear that LC/A-CPDY-H_(N)/A conjugates are more potent than theunliganded LC/A-H_(N)/A control molecule (labelled as LC/A-H_(N)/A).

FIG. 36—Kappa Receptor Activation Studies with a Range of DynorphinConjugates

Chinese hamster ovary (CHO) cells were transfected so that they expressthe OP2 receptor and SNAP-25. Said cells were used to measure cAMPdeletion that occurs when the receptor is activated with a dynorphinligand, using a FRET-based cAMP kit (LANCE kit from Perkin Elmer). Thetransfected cells were exposed to varying concentrations of dynorphinconjugates of the present invention for 2 hours. cAMP levels were thendetected by addition of a detection mix containing a fluorescentlylabelled cAMP tracer (Europium-streptavadi/biotin-cAMP) andfluorescently (Alexa) labelled anti-cAMP antibody and incubating at roomtemperature for 24 hours. Then samples are excited at 320 nM and emittedlight measured at 665 nM to determine cAMP levels. It is clear thatLC/A-CPDY-H_(N)/A conjugates are more potent than the unligandedLC/A-H_(N)/A control molecule (labelled as LC/A-H_(N)/A).

FIG. 37—Kappa Receptor Activation Studies with a Range of DynorphinConjugates

Chinese hamster ovary (CHO) cells were transfected so that they expressthe OP2 receptor (purchased from Perkin Elmer). Said cells weretransfected so they express SNAP-25 and used to measure cAMP deletionthat occurs when the receptor is activated with a dynorphin ligand,using a FRET-based cAMP kit (LANCE kit from Perkin Elmer). Thetransfected cells were exposed to varying concentrations of dynorphinconjugates of the present invention for 2 hours. cAMP levels were thendetected by addition of a detection mix containing a fluorescentlylabelled cAMP tracer (Europium-streptavadi/biotin-cAMP) andfluorescently (Alexa) labelled anti-cAMP antibody and incubating at roomtemperature for 24 hours. Then samples are excited at 320 nM and emittedlight measured at 665 nM to determine cAMP levels. It is clear from thefigure by the reduction in maximum cAMP that the OP2 receptor isactivated by LC/A-CPDY-H_(N)/A (labelled as CPDY/A), LC/B-CPDY-H_(N)/B(labelled as CPDY/B), LC/C—CPDY-H_(N)/C (labelled as CPDY/C), andLC/D-CPDY-H_(N)/D (labelled as CPDY/D). The concentration required toachieve 50% reduction in cAMP with LC/A-CPDY-H_(N)/A, LC/B-CPDY-H_(N)/B,LC/C-CPDY-H_(N)/C (labelled as CPDY/, and LC/D-CPDY-H_(N)/D is 10.47 nM,14.79 nM, 14.79 nM and 23.99 nM, respectively. Dynorphin peptidecontaining amino acids 1-17 of dynorphin A (labelled as dynorphin (1-17)was more potent than the fusions; 0.15 nm concentration required toachieve 50% reduction of cAMP.

FIG. 38—MrgX1 Receptor Activation Studies with BAM Conjugates

The ability of BAM conjugates of the invention to activate the MrgX1receptor in CHO cells was evaluated by measurement of the potency(pEC₅₀) and intrinsic efficacy (Emax) of ligands at the human MrgX1receptor. Receptor activation by an agonist to causes Gα_(g) proteinactivation resulting in Ca²⁺ release from intracellular stores that ismediated by the target enzyme phospholipase CB. The transient increasein intracellular Ca²⁺ was measured with a FlexStation3 microplate readerwith integrated fluid transfer. CHO cells that express the recombinanthuman MrgX1 receptor were incubated with the a FLIP R-Calcium-4 maskingdye and this Ca²⁺-4 dye formed a complex with Ca²⁺which fluoresces at525 nm following excitation at 485 nm allowing signal-detection. Aninhibitor of cell membrane anion exchanger, probenecid, was included inthe assay buffer to prevent outward transport or sequestration of dyemolecules. Following incubation with the dye, the cell plate was loadedonto to the FlexStation3 which transfers BAM conjugates (or referenceagonist BAMS-22) from a source plate into the microplate wellscontaining cells. The FlexStation 3 measured the fluorescent-emissionfrom the Calcium-4 dye and readouts were formed as calcium tracesdisplaying the magnitude of calcium flux as a result of MrgX1 receptoractivation. The data demonstrated the activation of the MrgX1 receptorby BAM conjugates of the invention.

FIG. 39—BAM Conjugate Efficacy in Capsaicin-Induced Thermal HyperalgesiaAssay

The ability of different BAM conjugates of the invention to inhibitcapsaicin-induced thermal hyperalgesia was evaluated. Intraplantarpretreatment of conjugates into Sprague-Dawley rats and 24 hours later0.3% capsaicin was injected and rats were put on 25° C. glass plate(rats contained in acrylic boxes, on 25° C. glass plate). Light beam(adjustable light Intensity) focused on the hind paw. Sensors detectedmovement of paw, stopping timer. Paw Withdrawal Latency is the timeneeded to remove the paw from the heat source (Cut-off of 20.48seconds). A reduction/inhibition of the paw withdrawal latency indicatesthat the test substance demonstrates an antinociceptive effect. The datademonstrated the antinociceptive effect of the BAM conjugates of thepresent invention.

FIG. 40—Conjugate Efficacy in Capsaicin-Induced Thermal HyperalgesiaAssay

The ability of different conjugates of the invention to inhibitcapsaicin-induced thermal hyperalgesia was evaluated. Intraplantarpretreatment of conjugates into Sprague-Dawley rats and 24 hours later0.3% capsaicin was injected and rats were put on 25° C. glass plate(rats contained in acrylic boxes, on 25° C. glass plate). Light beam(adjustable light Intensity) focused on the hind paw. Sensors detectedmovement of paw, stopping timer. Paw Withdrawal Latency is the timeneeded to remove the paw from the heat source (Cut-off of 20.48seconds). A reduction/inhibition of the paw withdrawal latency indicatesthat the test substance demonstrates an antinociceptive effect. The datademonstrated the antinociceptive effect of the conjugates of the presentis invention.

FIG. 41—Mu-Opiod Receptor (OPRM1) Binding Assay with β-EndorphinConjugates

Chinese hamster ovary (CHO) cells were stably transfected with the humanmu-opioid receptors (CHO-K1-OPRM1) and used in a radioligand competitionbinding assay using [3H]-DAMGO. The data demonstrated that theβ-endorphin fusion conjugates of the present invention having differentserotype backbones (i.e. A, B and D) demonstrated aconcentration-dependent and almost complete inhibition of the specificbinding of [3H]-DAMGO to the human mu-opioid receptors.

FIG. 42—Cleavage of SNARE Protein by β-Endorphin Conjugates in HumanSmall Cell Lung Carcinoma Cell Line NCI-H69

A SNAP-25 cleavage assay was developed using the human small cell lungcarcinoma cell line NCI-H69 expressing endogenous opiod receptors andthe activity of β-endorphin conjugates was assessed. The datademonstrated efficacy of the β-endorphin conjugates in SNARE cleavage.Maximum SNAP-25 cleavage achieved by CPBE fusion protein was 23% (ED₅₀38 nm).

FIG. 43—β-Endorphin Conjugate Efficacy in Capsaicin-Induced Paw GuardingAssay

The nociceptive flexion reflex (also known as paw guarding assay) is arapid withdrawal movement that constitutes a protective mechanismagainst possible limb damage. It can be quantified by assessment ofelectromyography (EMG) response in anesthetized rat as a result of lowdose capsaicin, electrical stimulation or the capsaicin-sensitizedelectrical response. Intraplantar pretreatment (24 hour) of testsubstance into 300-380 g male Sprague-Dawley rats. Induction of pawguarding in defined method is achieved by 0.006% capsaicin, 10 μl in PBS(7.5% DMSO), injected in 10 seconds. This produces a to robust reflexresponse from biceps feroris muscle. A reduction/inhibition of thenociceptive flexion reflex indicates that the test substancedemonstrates an antinociceptive effect. The paw guarding assay datademonstrated the antinociceptive effect of the β-endorphin conjugates ofthe present invention.

FIG. 44—β-Endorphin Conjugate Efficacy in Capsaicin-Induced ThermalHyperalgesia Assay

The ability of different β-endorphin conjugates of the invention toinhibit capsaicin-induced thermal hyperalgesia was evaluated.Intraplantar pretreatment of fusion proteins into Sprague-Dawley ratsand 24 hours later 0.3% capsaicin was injected and rats were put on 25°C. glass plate (rats contained in acrylic boxes, on 25° C. glass plate).Light beam (adjustable light Intensity) focused on the hind paw. Sensorsdetected movement of paw, stopping timer. Paw Withdrawal Latency is thetime needed to remove the paw from the heat source (Cut-off of 20.48seconds). A reduction/inhibition of the paw withdrawal latency indicatesthat the test substance demonstrates an antinociceptive effect. The datademonstrated the antinociceptive effect of the β-endorphin conjugates ofthe present invention.

FIG. 45—B₂ Receptor Activation Studies with Bradykinin Conjugates

Chinese hamster ovary (CHO) cells were stably transfected with the B₂receptor and used in a calcium fluorimetry assay measuring intracellularcalcium levels. The assay allowed the measurement of the potency (pEC₅₀)and intrinsic efficacy (E_(max)) of the bradykinin fusion protein. Thedata demonstrated that the bradykinin conjugates activated the B₂receptor and produced a dose dependent increase in intracellularcalcium.

FIG. 46—Bradykinin Conjugate Efficacy in Capsaicin-Induced Paw Guarding

The paw guarding assay data (conducted as described above for FIG. 43)demonstrated the antinociceptive effect of the bradykinin conjugates ofthe present invention.

FIG. 47—Bradykinin Conjugate Efficacy in Capsaicin-Induced ThermalHyperalgesia Assay

The thermal hyperalgesia assay data demonstrated (conducted as describedabove for FIG. 44) the antinociceptive effect of the bradykininconjugates of the present invention.

FIG. 48—B₂ Receptor Activation Studies with des-Arg⁹-BradykininConjugates

Chinese hamster ovary (CHO) cells were stably transfected with the B₁receptor and used in a calcium fluorimetry assay measuring intracellularcalcium levels. The assay allowed the measurement of the potency (pEC₅₀)and intrinsic efficacy (E_(max)) of conjugates having the des-Arg⁹-BKligand. The data demonstrated that the des-Arg⁹-BK fusion proteinactivated the B₁ receptor and produced a dose dependent increase inintracellular calcium.

DETAILED DESCRIPTION OF THE INVENTION

The use of an “agonist”, which would normally stimulate a biologicalprocess, particularly exocytosis (for example, an increase in cellularsecretion, or an up-regulation in membrane protein expression), is anexciting development in the technical field of re-targeted toxins.Furthermore, it is particularly surprising that an agonist may beemployed in a therapeutic composition to achieve a reduction orinhibition of a biological process that the agonist would normallystimulate.

The conjugates of the present invention represent a distinct sub-set oftoxin conjugates. In more detail, the conjugates of the presentinvention comprise TMs that have been selected on the basis of specificproperties rather than on the simple basis that they have acorresponding receptor on a pain-sensing target cell of interest.

Conventionally, an agonist has been considered any molecule that caneither increase or decrease activities within a cell, namely anymolecule that simply causes an alteration of cell activity. For example,the conventional meaning of an agonist would include: a chemicalsubstance capable of combining with a receptor on a cell and initiatinga reaction or activity, or a drug that induces an active response byactivating receptors, whether the response is an increase or decrease incellular activity.

However, for the purposes of this invention, an agonist is morespecifically defined as a molecule that is capable of stimulating theprocess of exocytic fusion in a pain-sensing target cell, which processis susceptible to inhibition by a protease (or fragment thereof) capableof cleaving a protein of the exocytic fusion apparatus in said targetcell.

Accordingly, the particular agonist definition of the present inventionwould exclude many molecules that would be conventionally considered asagonists. For example, nerve growth factor (NGF) is an agonist inrespect of its ability to promote neuronal differentiation via bindingto a TrkA receptor. However, NGF is not an agonist when assessed by theabove criteria because it is not a principal inducer of exocytic fusion.In addition, the process that NGF stimulates (i.e. cell differentiation)is not susceptible to inhibition by the protease activity of anon-cytotoxic toxin molecule.

In use, an agonist-containing conjugate of the present invention doesnot deactivate an agonist receptor on a pain-sensing target cell, butrather the protease activity of the conjugate serves to negate theagonist-mediated response.

Furthermore, once delivered to the cytosol of the pain-sensing targetcell, the protease component of a conjugate of the present inventioninhibits or blocks the action of all subsequent agonists capable ofcausing the same effect (i.e. increased exocytic fusion) in the sametarget cell. This is advantageous and means that the conjugates of thepresent invention have application in situations where multiple agonistsmay be responsible for causing the sensation of pain. Thus, whendesigning a conjugate of the present invention, the TM that is selectedfor delivery need not necessarily be the principal agonist involved incausing the sensation of pain.

Agonist-mediated delivery according to the present invention providesthe following significant advantage over previous non-cytotoxicprotease-containing therapeutics: use of an agonist may conferpreferential binding and/or internalisation properties on the conjugate.This, in turn, may result in more efficient delivery of the proteasecomponent to a pain-sensing target cell.

In addition, use of an agonist as a TM is self-limiting with respect toside-effects. In more detail, binding of an agonist to a pain-sensingtarget cell increases exocytic fusion, which may exacerbate thesensation of pain. However, the exocytic process that is stimulated byagonist binding is subsequently reduced or inhibited by the proteasecomponent of the conjugate.

In preferred embodiments of the invention, the TM is an agonist of theORL₁ receptor. The ORL₁ receptor is present on pain-sensing cells in thebody.

The ORL₁ receptor is a member of the G-protein-coupled class ofreceptors, and has a seven transmembrane domain structure. Theproperties of the ORL₁ receptor are discussed in detail in Mogil &Pasternak (2001), Pharmacological Reviews, Vol. 53, No. 3, pages381-415.

Throughout this specification, reference to the “ORL₁ receptor” embracesall members of the ORL₁ receptor family. Members of the ORL₁ receptorfamily typically have a seven transmembrane domain structure, and arecoupled to G-proteins of the G_(i) and G₀ families. A method fordetermining the G-protein-stimulating activity of ligands of the ORL₁receptor is given in Example 17. A method for measuring reduction incellular cAMP levels following ORL₁ activation is given in Example 16. Afurther characteristic of members of the ORL₁ receptor family is thatthey are typically able to bind nociceptin (the natural ligand of ORL₁).As an example, all alternative splice variants of the ORL₁ receptor, aremembers of the ORL₁ receptor family.

The conjugates of the present invention generally demonstrate a reducedbinding affinity (in the region of up to 100-fold) for nociceptivesensory afferent target cells when compared with the corresponding‘free’ TM. However, despite this observation, the conjugates of thepresent invention surprisingly demonstrate good efficacy. This can beattributed to two principal features. First, the non-cytotoxic proteasecomponent is catalytic—thus, the therapeutic effect of a few suchmolecules is rapidly amplified. Secondly, the receptors present on thenociceptive sensory afferents need only act as a gateway for entry ofthe therapeutic, and need not necessarily be stimulated to a levelrequired in order to achieve a ligand-receptor mediated pharmacologicalresponse. Accordingly, the conjugates of the present invention may beadministered at a dosage that is much lower that would be employed forother types of analgesic molecules such as NSAIDS, morphine, andgabapentin. The latter molecules are typically administered at highmicrogram to milligram (even up to hundreds of milligram) quantities,whereas the conjugates of the present invention may be administered atmuch lower dosages, typically at least 10-fold lower, and more typicallyat 100-fold lower.

In a particularly preferred embodiment of the invention, the TM of theconjugate is nociceptin—the natural ligand for the ORL₁ receptor.Nociceptin targets the ORL₁ receptor with high affinity.

Examples of Other Preferred TMs Include:

Code Sequence Ref. SEQ ID NO: Nociceptin 1-17 FGGFTGARKSARKLANQ [1] 1, 2Nociceptin 1-11 FGGFTGARKSA [1] 3, 4 Nociceptin [Y10]1-11 FGGFTGARKYA[1] 5, 6 Nociceptin [Y11]1-11 FGGFTGARKSY [1] 7, 8 Nociceptin [Y14]1-17FGGFTGARKSARKYANQ [1] 9, 10 Nociceptin 1-13 FGGFTGARKSARK [2] 11, 12Nociceptin [R14K15] FGGFTGARKSARKRKNQ [3,4] 13, 14 1-17 (also known as“variant” nociceptin) Nociceptin 1-13-NH₂ FGGFTGARKSARK-NH2 [5] 12Nociceptin (pNO₂)FGGFTGARKSARKLANQ [5] 2 Phe (p-NO₂) 1-17 LofentanilNon-peptide agonists [5] — Etorphine Non-peptide agonists [5] —Peptide agonist Peptide agonists from combinatorial  [6] —library approach [1] Mogil & Pasternak, 2001, Pharmacol. Rev., 53,381-415 [2] Maile et al., 2003, Neurosci. Lett., 350, 190-192 [3] Rizziet al., 2002, J. Pharmacol. Exp. Therap., 300, 57-63 [4] Okada et al.,2000, Biochem. Biophys. Res. Commun., 278, 493-498 [5] Zaveri, 2003,Life Sci., 73, 663-678. [6] Dooley et al., 1997, J Pharmacol Exp Ther.283(2), 735-41.

The TM preferably comprises a maximum of 50 amino acid residues, morepreferably a to maximum of 40 amino acid residues, particularlypreferably a maximum of 30 amino acid residues, and most preferably amaximum of 20 amino acid residues. For example, nociceptin is a 17 aminoacid residue peptide.

The above-identified “variant” TM demonstrates particularly good bindingaffinity (when compared with natural nociceptin) for nociceptive sensoryafferents. Generally speaking, a TM-containing conjugate willdemonstrate an approximate 100-fold reduction in binding abilityvis-à-vis the TM per se. The above-mentioned “variant” TM per sedemonstrates an approximate 3- to 10-fold increase in binding abilityfor a nociceptive sensory afferent vis-à-vis natural nociceptin. Thus, a“variant” TM-containing fusion might be expected to demonstrate anapproximate 10-fold reduction in binding ability for a nociceptivesensory afferent vis-à-vis ‘free’ nociceptin. However, the presentinventors have demonstrated that conjugates comprising said “variant” TMdemonstrate a binding ability that (most surprisingly) closely mirrorsthat of ‘free’ nociceptin—see FIG. 17.

In the context of the present invention, the term opiod or an agonist ofthe ORL₁ receptor (such as nociceptin, or any one of the peptides listedin the table above) embraces molecules having at least 70%, preferablyat least 80%, more preferably at least 90%, and most preferably at least95% amino acid sequence acid identity/homology with said opiod oragonist. The agonist homologues retain the agonist properties ofnociceptin at the ORL₁ receptor, which may be tested using the methodsprovided in Example 10. Similarly, an opioid homologue substantiallyretains the binding function of the opioid with which it shows highamino acid sequence identity/homology.

The invention also encompasses fragments, variants, and derivatives ofany one of the TMs described herein. These fragments, variants, andderivatives will substantially retain the properties that are ascribedto said TMs.

In addition to the above-mentioned opioid and non-opioid classes of TMs,a variety of other polypeptides are suitable for targeting theconjugates of the present invention to nociceptive sensory afferents(e.g. to nociceptors). In this regard, particular reference is made togalanin and derivatives of galanin. Galanin receptors are found pre- andpost-synaptically in DRGs (Liu & Hokfelt, (2002), Trends Pharm. Sci.,23(10), 468-74), and are enhanced in expression during neuropathic painstates. Proteinase-activated receptors (PARs) are also a preferred groupof TMs of the present invention, most particularly PAR-2. It is knownthat agonists of PAR-2 induce/elicit acute inflammation, in part via aneurogenic mechanism. PAR2 is expressed by primary spinal afferentneurons, and PAR2 agonists stimulate release of substance P(SP) andcalcitonin gene-related peptide (CGRP) in peripheral tissues. Anotherpreferred group of TMs of the present invention include bovine adrenalmedullary (BAM) peptides, bradykinin and/or substance P.

Another particularly preferred set of TMs of the present inventionincludes:

Ligand Reference Nociceptin Guerrini, et al., (1997) J. Med. Chem., 40,pp. 1789-1793 β-endorphin Blanc, et al., (1983) J. Biol. Chem., 258(13),pp. 8277-8284 Endomorphin-1; Zadina, et al., (1997). Nature, 386, pp.499-502 Endomorphin-2 Dynorphin Fields & Basbaum (2002) Chapter 11, InThe Textbook of Pain, Wall & Melzack eds. Met-enkephalin Fields &Basbaum (2002) Chapter 11, In The Textbook of Pain, Wall & Melzack eds.Leu-enkephalin Fields & Basbaum (2002) Chapter 11, In The Textbook ofPain, Wall & Melzack eds. Galanin Xu et al., (2000) Neuropeptides, 34(3&4), 137-147 PAR-2 peptide Vergnolle et al., (2001) Nat. Med., 7(7),821-826

In a preferred embodiment of the invention, the target for the TM isselected from the group consisting of: Mrg receptors such as MrgX1,opiod receptors such as OPRD1 and/or OPRM1, BDKRB1 and/or BDKRB2,Tachykinin receptors such as TACR1, TACR2 and/or TACR3, Kappa receptor(OPRK1) and/or ORL₁ receptor.

In one embodiment, the TM is a molecule that binds (preferably thatspecifically binds) to one or more of the above-mentioned receptors. Forexample, the TM is an “agonist” of one or more of the above-mentionedreceptors. The term “agonist” in this context is defined as above.

In one embodiment, the TM comprises or consists of a BAM peptide.Full-length BAM is a 22 amino acid peptide, abbreviated herein asBAM1-22 (represented by SEQ ID NO: 120). In one embodiment, the BAM TMof the invention comprises or consists of a 15 amino acid fragment offull-length BAM peptide and is referred to herein as BAMS-22(represented by SEQ ID NO: 121). In one embodiment, said BAM peptidesbind (preferably specifically bind) to Mrg receptors such as MrgX1.

In one embodiment, the TM comprises or consists of a β-endorphinpeptide. β-endorphin is a 31 amino acid peptide (represented by SEQ IDNO: 126). In one embodiment, said β-endorphin peptide binds (preferablyspecifically binds) opioid receptors such as OPRD1 and/or OPRM1.

In one embodiment, the TM comprises or consists of a bradykinin peptide.bradykinin is a 9 amino acid peptide (represented by SEQ ID NO: 129). Inone embodiment, said bradykinin peptide binds (preferably specificallybinds) bradykinin target receptors BDKRB1 and/or BDKRB2.

In one embodiment, the TM comprises or consists of a des-Arg⁹-BK ligand(represented by SEQ ID NO: 130). The des-Arg9-Bradykinin ligand differsfrom bradykinin ligand by the removal of an arginine residue from theC-terminus. In one embodiment, said des-Arg⁹-BK ligand binds (preferablyspecifically binds) bradykinin target receptors BDKRB1 and/or BDKRB2.

In one embodiment, the TM comprises or consists of a substance Ppeptide. Full length substance P is an 11 amino acid peptide(represented by SEQ ID NO: 134). In one embodiment, the TM comprises orconsists of a substance P analogue, such as the analogue referred toherein as ‘S6’ (represented by SEQ ID NO: 135). In one embodiment, saidsubstance P peptide, or analogue thereof binds (preferably specificallybinds) to Tachykinin receptors such as TACR1, TACR2 and/or TACR3.

In one embodiment, the TM comprises or consists of a dynorphin peptide.The sequence of dynorphin is represented by SEQ ID NO: 101. In oneembodiment, said dynorphin peptide binds (preferably specifically binds)Kappa receptor (OPRK1).

The invention also encompasses fragments, variants, and derivatives andanalogues of the above-mentioned TMs. These fragments, variants, andderivatives and analogues substantially retain the properties that areascribed to said TM. For example, the fragments, variants, andderivatives may retain the ability to bind to their respectivereceptor(s). By way of example, reference is made to the above-mentionedBAMS-22 fragment of the full length BAM1-22 ™ as well as the substance Panalogue S6.

In one embodiment, the TM comprises or consist of an amino acid sequencehaving at least 70%, preferably at least 80% (such as at least 82, 84,85, 86, 88 or 89%), more preferably at least 90% (such as at least 91,92, 93 or 94%), and most preferably at least 95% (such as at least 96,97, 98, 99 or 100%) amino acid sequence acid identity to SEQ ID NO: 2,4, 12, 101, 120, 121, 126, 129, 130, 134 and/or 135.

In one embodiment, the TM comprises or consist of an amino acid sequencehaving at least 70%, preferably at least 80% (such as at least 82, 84,85, 86, 88 or 89%), more preferably at least 90% (such as at least 91,92, 93 or 94%), and most preferably at least 95% (such as at least 96,97, 98, 99 or 100%) amino acid sequence acid identity to SEQ ID NO: 120,121, 126, 129, 130, 134 and/or 135.

In one embodiment, the Targeting Moiety comprises or consists of anamino acid sequence according to SEQ ID NO: 120, 121, 126, 129, 130, 134and/or 135 or a fragment comprising or consisting of at least 16 (suchas at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15) contiguous aminoacid residues thereof, or a variant amino acid sequence of said SEQ IDNO: 120, 121, 126, 129, 130, 134 and/or 135 or said fragment having amaximum of 6 (such as a maximum of 5, 4, 3, 2 or 1) conservative aminoacid substitutions.

The agonist properties of a TM can be confirmed using the methodsdescribed in Example 1. These methods are based on previous experiments(see Inoue et al. (1998) Proc. Natl. Acad. Sci., 95, 10949-10953), whichconfirm that the natural agonist of the ORL₁ receptor, nociceptin,causes the induction of substance P release from nociceptive primaryafferent neurons. This is supported by the facts that:

-   -   the nociceptin-induced responses are abolished by specific NK1        receptor (the substance P receptor) antagonists; and    -   pre-treatment of the cells with capsaicin (which depletes        substance P from small diameter primary afferent neurons)        attenuates the nociceptin-induced responses.

Similarly, Inoue et al. confirm that an intraplantar injection ofbotulinum neurotoxin type A abolishes the nociceptin-induced responses.Since it is known that BoNT inhibits the release of substance P fromprimary afferent neurons (Welch et al., (2000), Toxicon, 38, 245-258),this confirms the link between nociceptin-ORL₁ interaction andsubsequent release of substance P.

Thus, a TM can be said to have agonist activity at the ORL₁ receptor ifthe TM causes an induction in the release of substance P from anociceptive sensory afferent neuron (see Example 1).

In another embodiment, opioids represent a preferred group of TMs of thepresent invention. Within this family of peptides is includedenkephalins (met and leu), endomorphins 1 and 2, β-endorphin anddynorphin. Opioid peptides are frequently used in the clinic to modifythe activity to nociceptors, and other cells involved in the painresponse. As exemplified by the three-step World Health OrganisationAnalgesic Ladder, opioids have entry points into the pharmacologicaltreatment of chronic cancer and non-cancer pain at all three stages,underlining their importance to the treatment of pain. Reference toopioids embraces fragments, variants and derivatives thereof, whichretain the ability to bind to nociceptive sensory afferents.

The protease of the present invention embraces all naturally-occurringnon-cytotoxic proteases that are capable of cleaving one or moreproteins of the exocytic fusion apparatus in eukaryotic cells.

The protease of the present invention is preferably a bacterialprotease.

More preferably, the bacterial protease is selected from the generaClostridium or Neisseria (e.g. a clostridial L-chain, or a neisserialIgA protease preferably from N. gonorrhoeae).

The present invention also embraces modified non-cytotoxic proteases,which include amino acid sequences that do not occur in nature and/orsynthetic amino acid residues, so long as the modified proteases stilldemonstrate the above-mentioned protease activity.

The protease of the present invention preferably demonstrates a serineor metalloprotease activity (e.g. endopeptidase activity). The proteaseis preferably specific for a SNARE protein (e.g. SNAP-25,synaptobrevin/VAMP, or syntaxin).

Particular mention is made to the protease domains of neurotoxins, forexample the protease domains of bacterial neurotoxins. Thus, the presentinvention embraces the use of neurotoxin domains, which occur in nature,as well as recombinantly prepared versions of said naturally-occurringneurotoxins.

Exemplary neurotoxins are produced by clostridia, and the termclostridial neurotoxin embraces neurotoxins produced by C. tetani(TeNT), and by C. botulinum (BoNT) serotypes A-G, as well as the closelyrelated BoNT-like neurotoxins produced by C. baratii and C. butyricum.The above-mentioned abbreviations are used throughout the presentspecification. For example, the nomenclature BoNT/A denotes the sourceof neurotoxin as BoNT (serotype A). Corresponding nomenclature appliesto other BoNT serotypes.

The term L-chain fragment means a component of the L-chain of aneurotoxin, which fragment demonstrates a metalloprotease activity andis capable of proteolytically cleaving a vesicle and/or plasma membraneassociated protein involved in cellular exocytosis.

A Translocation Domain is a molecule that enables translocation of aprotease (or fragment thereof) into a pain-sensing target cell such thata functional expression of protease activity occurs within the cytosolof the target cell. Whether any molecule (e.g. a protein or peptide)possesses the requisite translocation function of the present inventionmay be confirmed by any one of a number of conventional assays.

For example, Shone C. (1987) describes an in vitro assay employingliposomes, which are challenged with a test molecule. Presence of therequisite translocation function is confirmed by release from theliposomes of K⁺ and/or labelled NAD, which may be readily monitored (seeShone C. (1987) Eur. J. Biochem; vol. 167(1): pp. 175-180).

A further example is provided by Blaustein R. (1987), which describes asimple in vitro assay employing planar phospholipid bilayer membranes.The membranes are challenged with a test molecule and the requisitetranslocation function is confirmed by an increase in conductance acrosssaid membranes (see Blaustein (1987) FEBS Letts; vol. 226, no. 1: pp.115-120).

Additional methodology to enable assessment of membrane fusion and thusidentification of Translocation Domains suitable for use in the presentinvention are provided by Methods in Enzymology, Vols. 220 and 221,Membrane Fusion Techniques, Parts A and B, Academic Press 1993.

The Translocation Domain is preferably capable of formation ofion-permeable pores in is lipid membranes under conditions of low pH.Preferably, it has been found to use only those portions of the proteinmolecule capable of pore-formation within the endosomal membrane.

The Translocation Domain may be obtained from a microbial proteinsource, in particular from a bacterial or viral protein source. Hence,in one embodiment, the Translocation Domain is a translocating domain ofan enzyme, such as a bacterial toxin or viral protein.

It is well documented that certain domains of bacterial toxin moleculesare capable of forming such pores. It is also known that certaintranslocation domains of virally expressed membrane fusion proteins arecapable of forming such pores. Such domains may be employed in thepresent invention.

The Translocation Domain may be of a clostridial origin, namely theH_(N) domain (or a functional component thereof). H_(N) means a portionor fragment of the H-chain of a clostridial neurotoxin approximatelyequivalent to the amino-terminal half of the H-chain, or the domaincorresponding to that fragment in the intact H-chain. Examples ofsuitable clostridial Translocation Domains include:

-   -   Botulinum type A neurotoxin—amino acid residues (449-871)    -   Botulinum type B neurotoxin—amino acid residues (441-858)    -   Botulinum type C neurotoxin—amino acid residues (442-866)    -   Botulinum type D neurotoxin—amino acid residues (446-862)    -   Botulinum type E neurotoxin—amino acid residues (423-845)    -   Botulinum type F neurotoxin—amino acid residues (440-864)    -   Botulinum type G neurotoxin—amino acid residues (442-863)    -   Tetanus neurotoxin—amino acid residues (458-879)

For further details on the genetic basis of toxin production inClostridium botulinum and C. tetani, we refer to Henderson et al. (1997)in The Clostridia: Molecular Biology and Pathogenesis, Academic press.

The term H_(N) embraces naturally-occurring neurotoxin H_(N) portions,and modified H_(N) portions having amino acid sequences that do notoccur in nature and/or synthetic amino acid residues, so long as themodified H_(N) portions still demonstrate the above-mentionedtranslocation function.

Alternatively, the Translocation Domain may be of a non-clostridialorigin (see table below). Examples of non-clostridial TranslocationDomain origins include, but are not restricted to, the translocationdomain of diphtheria toxin [O'Keefe et al., Proc. Natl. Acad. Sci. USA(1992) 89, 6202-6206; Silverman et al., J. Biol. Chem. (1993) 269,22524-22532; and London, E. (1992) Biochem. Biophys. Acta., 1112, pp.25-51], the translocation domain of Pseudomonas exotoxin type A [Prioret al. Biochemistry (1992) 31, 3555-3559], the translocation domains ofanthrax toxin [Blanke et al. Proc. Natl. Acad. Sci. USA (1996) 93,8437-8442], a variety of fusogenic or hydrophobic peptides oftranslocating function [Plank et al. J. Biol. Chem. (1994) 269,12918-12924; and Wagner et al (1992) PNAS, 89, pp. 7934-7938], andamphiphilic peptides [Murata et al (1992) Biochem., 31, pp. 1986-1992].The Translocation Domain may mirror the Translocation Domain present ina naturally-occurring protein, or may include amino acid variations solong as the variations do not destroy the translocating ability of theTranslocation Domain.

Particular examples of viral Translocation Domains suitable for use inthe present invention include certain translocating domains of virallyexpressed membrane fusion proteins. For example, Wagner et al. (1992)and Murata et al. (1992) describe the translocation (i.e. membranefusion and vesiculation) function of a number of fusogenic andamphiphilic peptides derived from the N-terminal region of influenzavirus haemagglutinin. Other virally expressed membrane fusion proteinsknown to have the desired translocating activity are a translocatingdomain of a fusogenic peptide of Semliki Forest Virus (SFV), atranslocating domain of vesicular stomatitis virus (VSV) to glycoproteinG, a translocating domain of SER virus F protein and a translocatingdomain of Foamy virus envelope glycoprotein. Virally encoded “spikeproteins” have particular application in the context of the presentinvention, for example, the E1 protein of SFV and the G protein of VSV.

Use of the Translocation Domains (listed below) includes use of sequencevariants thereof. A variant may comprise one or more conservativenucleic acid substitutions and/or nucleic acid deletions or insertions,with the proviso that the variant possesses the requisite translocatingfunction. A variant may also comprise one or more amino acidsubstitutions and/or amino acid deletions or insertions, so long as thevariant possesses the requisite translocating function.

Translocation Amino acid Domain source residues References Diphtheriatoxin 194-380 Silverman et al., 1994, J. Biol. Chem. 269, 22524-22532London E., 1992, Biochem. Biophys. Acta., 1113, 25-51 Domain II of405-613 Prior et al., 1992, pseudomonas Biochemistry 31, exotoxin3555-3559 Kihara & Pastan, 1994, Bioconj Chem. 5, 532-538 Influenzavirus GLFGAIAGFIENGWE Plank et al., haemagglutinin GMIDGWYG (SEQ ID1994, J. Biol. Chem. NO: 170), and 269, 12918-12924 Variants thereofWagner et al., 1992, PNAS, 89, 7934-7938 Murata et al., 1992,Biochemistry 31, 1986-1992 Semliki Forest virus Translocation domainKielian et al., fusogenic protein 1996, J Cell Biol. 134(4), 863-872Vesicular Stomatitis 118-139 Yao et al., 2003, virus glycoprotein GVirology 310(2), 319-332 SER virus F protein Translocation domain Sethet al., 2003, J Virol 77(11) 6520-6527 Foamy virus Translocation domainPicard-Maureau et al., envelope 2003, J Virol. glycoprotein 77(8),4722-4730

Once a potential receptor agonist (e.g. an ORL1 agonist) has beenidentified, one or more of the following optional steps may be carriedout:

-   -   (A) confirming that the putative agonist molecule or agonist is        capable of being combined with a non-cytotoxic protease (or a        fragment thereof) and optionally a Translocation Domain to form        a conjugate of the present invention; and/or    -   (B) confirming that said putative agonist molecule or agonist        binds to the receptor on the pain-sensing target cell, which        receptor is susceptible to receptor-mediated endocytosis; and/or    -   (C) confirming that said putative agonist molecule or agonist is        able to deliver a non-cytotoxic protease (or fragment thereof)        into the cytosol of a pain-sensing target cell.

The above steps (A)-(C) may be confirmed by routine tests that would bereadily available to a skilled person.

For example, step (A) may be performed by a simple chemical conjugationexperiment using conventional conjugation reagents and/or linkermolecules, followed by native polyacrylamide gel electrophoresis toconfirm that a conjugate of the present invention is formed that has theanticipated molecular weight. The conjugate components are typicallylinked together (optionally via linker molecules) by covalent bonds.

For example, step (B) may be performed by any one of a range ofmethodologies for assessment of binding of a ligand. Standard text, forexample “Receptor-Ligand Interactions. A Practical Approach. Ed. E. C.Hulme, IRL Press, 1992” are available that describe such approaches indetail. In brief, the agonist or putative agonist molecule is labelled(for example, with 125-iodine) and applied to a cell preparation invitro in the presence of an excess of unlabelled agonist. The purpose ofthe unlabelled material is to saturate any non-specific binding sites.The agonist is incubated with the cell preparation for sufficient timeto achieve equilibrium, and the amount of label bound to the cellsassessed by measuring cell associated radioactivity, for example byscintillation or gamma counting.

A further example involves gold-labelling of the agonist (or putativeagonist), followed by the use of electron microscopy to monitor thecellular transport progress of the labelled agonist [see the basicmethodology described by Rabinowitz S. (1992); J. Cell. Biol. 116 (1):pp. 95-112; and that described by van Deurs (1986); J. Cell. Biol. 102:pp. 37-47].

For example, step (C) may be performed by contacting the conjugateprepared in step (A) with a suitable target cell and assessing cleavageof the substrate. This is performed by extraction of the SNARE proteins,followed by Western blotting of SDS-PAGE-separated samples. Cleavage ofsubstrate is indicative of delivery of the protease into the targetcell. In this regard, cleavage may be monitored by disappearance ofsubstrate and/or appearance of cleavage product. A particularly usefulantibody that selectively binds to the cleaved substrate product isdescribed in WO95/33850.

Preparation of a conjugate according to the present invention is nowdiscussed.

It is known in the art that the H_(C) portion of a neurotoxin moleculecan be removed from the other portion of the H-chain, known as H_(N),such that the H_(N) fragment remains disulphide linked to the L-chain ofthe neurotoxin providing a fragment known as LH_(N). Thus, in oneembodiment of the present invention the LH_(N) fragment of a neurotoxinis covalently linked, using linkages which may include one or morespacer regions, to a TM.

In another embodiment of the invention, the H_(C) domain of a neurotoxinis mutated, blocked or modified, e.g. by chemical modification, toreduce or preferably incapacitate its ability to bind the neurotoxin toreceptors at the neuromuscular junction. This modified neurotoxin isthen covalently linked, using linkages which may include one or morespacer regions, to a TM.

In another embodiment of the invention, the H-chain of a neurotoxin, inwhich the H_(C) domain is mutated, blocked or modified, e.g. by chemicalmodification, to reduce or preferably incapacitate its native bindingability, is combined with the L-chain of a different neurotoxin, oranother protease capable of cleaving a protein of the exocytic fusionapparatus (e.g. IgA protease of N. gonorrhoeae). This hybrid, modifiedneurotoxin is then covalently linked, using linkages which may includeone or more spacer regions, to a TM.

In another embodiment of the invention, the H_(N) domain of a neurotoxinis combined with is the L-chain of a different neurotoxin, or anotherprotease capable of cleaving a protein of the exocytic fusion apparatus(e.g. IgA protease of N. gonorrhoeae). This hybrid is then covalentlylinked, using linkages which may include one or more spacer regions, toa TM.

In another embodiment of the invention, the protease (for example theL-chain component of a neurotoxin) is covalently linked, using linkagesthat may include one or more spacer regions, to a TM that can alsoeffect the internalisation of the protease into the cytoplasm of therelevant target cell(s).

In another embodiment of the invention, the protease (for example theL-chain component of a neurotoxin) is covalently linked, using linkageswhich may include one or more spacer regions, to a translocation domainto effect transport of the protease fragment into the cytosol.

In use, the domains of a conjugate according to the present inventionare associated with each other. In one embodiment, two or more of thedomains may be joined together either directly (e.g. by a covalentlinkage), or via a linker molecule.

A variety of different linker/spacer molecules may be employed in any ofthe fusion proteins of the present invention. Examples of such spacermolecules include those illustrated in FIGS. 31 and 32. Particularmention here is made to GS15, GS20, GS25, and Hx27—see FIGS. 31 and 32.

The present inventors have unexpectedly found that non-cytotoxicprotease-TM conjugates (eg. CPNv/A) may demonstrate an improved bindingactivity for nociceptive sensory afferents when the size of the spaceris selected so that (in use) the TM (preferably the C-terminus thereof)and the translocation domain (preferably the N-terminus thereof) areseparated from one another by 40-105 angstroms, preferably by 50-100angstroms, and more preferably by 50-90 angstroms. In anotherembodiment, the preferred spacers have an amino acid sequence of 11-29amino acid residues, preferably 15-27 amino acid residues, and morepreferably 20-27 amino acid residues. Suitable spacers may be routinelyidentified and obtained according to Crasto, C. J. and Feng, J. A.(2000) May, 13 (5), pp. 309-312—see also the website having a URL endingin: fccc./edu/research/labs/feng/limker.html.

Conjugation techniques suitable for use in the present invention havebeen well documented and are routine for a person skilled in the art.

The methodology involved in coupling two protein molecules (A and B)together is simple, and is achieved through the use of a cross-linkingagent (also known as a chemical coupling agent). For example, moleculesA and B are separately contacted with a cross-linking agent, whichchemically modifies a specific surface group on each of molecules A andB thereby forming derivatised molecules A′ and B′. The modified surfacegroup on molecule A′ is capable of covalently bonding with the modifiedsurface group on molecule B′. Thus, the coupling reaction is completedby mixing together the two protein molecules A′ and B′.

Chemical conjugation is illustrated by reference to the followingembodiments, where P=non-cytotoxic protease component, T=translocationcomponent, and TM=targeting moiety.

In one embodiment, a single chain P-T is prepared, which is thenconjugated to a TM. In another embodiment, a single chain TM-T (or T-TM)is prepared, which is then conjugated to a P. In a further embodiment, asingle chain P-TM (or TM-P) is prepared, which is then conjugated to aT. Another particularly preferred conjugate has the structure P-TM-T(with an optional protease cleavage site between P and TM).

Where the T and P components are prepared as a single chain polypeptide,a protease cleavage site is typically included between said components.Any protease cleavage site may be employed in this regard.

In an alternative embodiment, the three components may be simultaneouslyor sequentially conjugated together. Thus, the conjugation may be a one-or two-step process, and may include one or more different couplingagents.

In another embodiment of the present invention, the TM is either N- orC-terminally located with respect to the conjugate. In other words, inone embodiment the TM is not located between the non-cytotoxic proteaseand translocation domain components of the non-cytotoxic proteinconjugate.

In one embodiment, the invention provides a non-cytotoxic proteinconjugate comprising (or consisting of) an amino acid sequence having atleast 80% (such as at least 85, 90, 92, 94, 95, 96, 97, 98, 99 or 100%)sequence identity to the amino acid sequence of SEQ ID NOs: 50, 110,111, 112, 113, 114, 115, 116, 117, 118, 119, 123, 124, 125, 127, 128,132, 133, 136, 137, 138, 141, 142, 143, 144, 145, 146, 147, 148, 149,150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,164, 165, 166, 167, 168, and/or 169.

In one embodiment, the invention provides a non-cytotoxic proteinconjugate comprising comprising (or consisting of) an amino acidsequence having at least 80% (such as at least 85, 90, 92, 94, 95, 96,97, 98, 99 or 100%) sequence identity to the amino acid sequence of SEQID NOs: 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 141, 152, 153,154, 155, 156, 157, 158, 159, 160, 161, and/or 162.

In one embodiment, the invention provides a non-cytotoxic proteinconjugate comprising (or consisting of) an amino acid sequence having atleast 80% (such as at least 85, 90, 92, 94, 95, 96, 97, 98, 99 or 100%)sequence identity to the amino acid sequence of SEQ ID NOs: 123, 124,125, 144, 145 and/or 146.

In one embodiment, the invention provides a non-cytotoxic proteinconjugate comprising (or consisting of) an amino acid sequence having atleast 80% (such as at least 85, 90, 92, 94, 95, 96, 97, 98, 99 or 100%)sequence identity to the amino acid sequence of SEQ ID Nos: 50, 127,128, 147, 148, 149 and/or 150.

In one embodiment, the invention provides a non-cytotoxic proteinconjugate comprising (or consisting of) an amino acid sequence having atleast 80% (such as at least 85, 90, 92, 94, 95, 96, 97, 98, 99 or 100%)sequence identity to the amino acid sequence of SEQ ID NOs: 132, 133and/or 151.

In one embodiment, the invention provides a non-cytotoxic proteinconjugate comprising (or consisting of) an amino acid sequence having atleast 80% (such as at least 85, 90, 92, 94, 95, 96, 97, 98, 99 or 100%)sequence identity to the amino acid sequence of SEQ ID NO: 136 and/or169.

In one embodiment, the invention provides a non-cytotoxic proteinconjugate comprising (or consisting of) an amino acid sequence having atleast 80% (such as at least 85, 90, 92, 94, 95, 96, 97, 98, 99 or 100%)sequence identity to the amino acid sequence of SEQ ID NOs: 137, 138,142, 143, 163, 164, 165, 166, 167 and/or 168.

Chemical coupling agents and cross-linking agents have been commerciallyavailable for many years.

Example 5 of the present invention describes in detail the use of onesuch coupling agent, namely SPDP, to chemically couple two proteinmolecules (nociceptin, and the LH_(N) of botulinum neurotoxin). The twomolecules are separately contacted with SPDP, and then mixed together toallow covalent conjugation.

The conjugate described in Example 6 confirms that another couplingagent, PDPH/EDAC, or Traut's reagent, may be employed as an alternativecoupling agent to SPDP.

SPDP and Traut's reagent are popular and well-documented coupling agentsin the technical field of protein conjugation chemistry and arepresented here simply as two examples of a well known class of compoundsthat may be employed to covalently link together the Targeting Moietycomponent and the clostridial neurotoxin component of the conjugate ofthe present invention. Other suitable agents include SMPB, SMCC(succinimidyl 4-(N-maleimidomethyl)cyclohexan-1-carboxylate), andLC-SPDP.

In more detail, commercially available members of the well-knowncoupling agents may be used for conjugation purposes to produce aconjugate of the invention. Details of such agents can be found in thefollowing publications:

-   Hermanson, G. T. (1996), Bioconjugate techniques, Academic Press;-   Wong, S. S. (1991), Chemistry of protein conjugation and    cross-linking, CRC Press;-   Thorpe et al (1987), Cancer Res, 1987, 47, 5924-31. This paper    describes the use of SMBT (sodium    S-4-succinimidyloxycarbonyl-alpha-methyl benzyl thiosulfate) and    SMPT    (4-succinimidyloxycarbonyl-alpha-methyl-alpha(2-pyridyldithio)toluene);    and-   Peeters et al (1989), J Immunol Methods. 1989, 120, 133-43. This    paper describes the use of 4 coupling reagents, MHS (succinimidyl    6-(N-maleimido)-n-hexanoate), SMCC (succinimidyl    4-(N-maleimidomethyl)-cyclohexane-1-carboxylate), MBS (succinimidyl    m-maleimidobenzoate), and SPDP.

The conjugates according to the present invention may also be preparedrecombinantly, as detailed in Examples 9 to 12.

In one embodiment, the preparation of a recombinant conjugate involvesarrangement of the coding sequences of a selected TM, a selectednon-cytotoxic protease component, and a translocation component (in anyorder) in a single genetic construct. These coding sequences may bearranged in-frame so that subsequent transcription and translation iscontinuous through both coding sequences and results in a fusionprotein. All constructs would have a 5′ ATG codon to encode anN-terminal methionine, and a C-terminal translational stop codon.

Thus, the recombinant preparation method results in the generation of asingle chain polypeptide. In order to activate this polypeptide, aprotease cleavage site is present between the non-cytotoxic proteasecomponent and the translocation component. Cleavage of this sitegenerates a di-chain polypeptide in which the protease and totranslocation domains are linked together by way of a covalent bond,preferably a disulphide bond. In this regard, any protease cleavage sitemay be employed.

In the single polypeptide aspect of the present invention, the TM may beN- or C-terminally located with respect to the fusion protein. In otherwords, in one embodiment is the TM is not located between thenon-cytotoxic protease and translocation domain components of thesingle-chain polypeptide fusion protein.

In another embodiment, the TM is located between the non-cytotoxicprotease and translocation domain components of the single-chainpolypeptide fusion protein.

In one embodiment, an L-chain of a clostridial neurotoxin or anotherprotease capable of cleaving a protein of the exocytic fusion apparatus(e.g. an IgA protease), or a fragment/variant thereof, may be expressedrecombinantly as a fusion protein with a TM, which TM can also effectthe internalisation of the L-chain component into the cytoplasm of therelevant target cell(s) responsible for secretion. Alternatively, thefusion protein may further comprise a Translocation Domain. Theexpressed fusion protein may include one or more spacer regions.

By way of example, the following information is required to produce,recombinantly, an agent of the present invention:

-   -   (I) DNA sequence data relating to a selected TM;    -   (II) DNA sequence data relating to the protease component;    -   (III) DNA sequence data relating to the translocation domain;        and    -   (IV) a protocol to permit construction and expression of the        construct comprising (I), (II) and (III).

All of the above basic information (I)-(IV) are either readilyavailable, or are readily determinable by conventional methods. Forexample, both WO98/07864 and WO99/17806 exemplify recombinant technologysuitable for use in the present application.

In addition, methods for the construction and expression of theconstructs of the present invention may employ information from thefollowing references and others:

-   Lorberboum-Galski, H., FitzGerald, D., Chaudhary, V., Adhya, S.,    Pastan, I. (1988), Cytotoxic activity of an interleukin    2-Pseudomonas exotoxin chimeric protein produced in Escherichia    coli. Proc. Natl. Acad. Sci. USA, 85(6):1922-6;-   Murphy, J. R. (1988), Diphtheria-related peptide hormone gene    fusions: a molecular genetic approach to chimeric toxin development.    Cancer Treat. Res.; 37:123-40;-   Williams, D. P., Parker, K., Bacha, P., Bishai, W., Borowski, M.,    Genbauffe, F., Strom, T. B., Murphy, J. R. (1987), Diphtheria toxin    receptor binding domain substitution with interleukin-2: genetic    construction and properties of a diphtheria toxin-related    interleukin-2 fusion protein. Protein Eng; 1(6):493-8;-   Arora, N., Williamson, L. C., Leppla, S. H., Halpern, J. L. (1994),    Cytotoxic effects of a chimeric protein consisting of tetanus toxin    light chain and anthrax toxin lethal factor in non-neuronal cells J.    Biol. Chem., 269(42):26165-71;-   Brinkmann, U., Reiter, Y., Jung, S. H., Lee, B., Pastan, I. (1993),    A recombinant immunotoxin containing a disulphide-stabilized Fv    fragment. Proc. Natl. Acad. Sci. USA, 90(16):7538-42; and-   O'Hare, M., Brown, A. N., Hussain, K., Gebhardt, A., Watson, G.,    Roberts, L. M., Vitetta, E. S., Thorpe, P. E., Lord, J. M. (1990),    Cytotoxicity of a recombinant ricin-A-chain fusion protein    containing a proteolytically-cleavable spacer sequence. FEBS Lett    October 29; 273 (1-2):200-4.

Suitable clostridial neurotoxin sequence information relating to L- andLH_(N)-chains may be obtained from, for example, Kurazono, H. (1992) J.Biol. Chem., vol. 267, No. 21, pp. 14721-14729; and Popoff, M. R., andMarvaud, J.-C. (1999) The Comprehensive Sourcebook of Bacterial ProteinToxins, 2nd edition (ed. Alouf, J. E., and Freer, J. H.), AcademicPress, pp. 174-201.

All of the aforementioned publications are hereby incorporated into thepresent specification by reference thereto.

Similarly, suitable TM sequence data are widely available in the art.Alternatively, any necessary sequence data may be obtained by techniqueswhich are well-known to the skilled person.

For example, DNA encoding the TM component may be cloned from a sourceorganism by screening a cDNA library for the correct coding region (forexample by using specific oligonucleotides based on the known sequenceinformation to probe the library), isolating the TM DNA, sequencing thisDNA for confirmation purposes, and then placing the isolated DNA in anappropriate expression vector for expression in the chosen host.

As an alternative to isolation of the sequence from a library, theavailable sequence information may be employed to prepare specificprimers for use in PCR, whereby the coding sequence is then amplifieddirectly from the source material and, by suitable use of primers, maybe cloned directly into an expression vector.

Another alternative method for isolation of the coding sequence is touse the existing sequence information and synthesise a copy, possiblyincorporating alterations, using DNA synthesis technology. For example,DNA sequence data may be generated from existing protein and/or RNAsequence information. Using DNA synthesis technology to do this (and thealternative described above) enables the codon bias of the codingsequence to be modified to be optimal for the chosen expression host.This may give rise to superior expression levels of the fusion protein.

Optimisation of the codon bias for the expression host may be applied tothe DNA sequences encoding the TM and clostridial components of theconstruct. Optimisation of the codon bias is possible by application ofthe protein sequence into freely available DNA/protein databasesoftware, e.g. programs available from Genetics Computer Group, Inc.

Having prepared a conjugate of the invention, it is a matter of routineto confirm that the various domains have retained their specifiedfunction.

Protease function after conjugation may be tested by using, for example,any one of the following routine tests:

SNAP-25 (or synaptobrevin, or syntaxin) may be challenged with aconjugate to be tested, and then analysed by SDS-PAGE peptide separationtechniques. Subsequent detection of peptides (e.g. by silver staining)having molecular weights corresponding to the cleaved products ofSNAP-25 (or other component of the neurosecretory machinery) is wouldconfirm the presence of a functional L-chain.

As a further alternative, the conjugate may be tested by assaying forSNAP-25 (or synaptobrevin, or syntaxin) cleavage products viaantibody-specific binding (see WO95/33850). In more detail, a specificantibody is employed for detecting cleavage of SNAP-25. Since theantibody recognises cleaved SNAP-25, but not uncleaved SNAP-25,identification of the cleaved product by the antibody confirms thepresence of L-chain proteolytic function. By way of exemplification,such a method is described in Examples 2 and 3 of WO96/33273.

Translocation component function after conjugation may be tested using,for example, any one of the following routine tests:

Suitable methods are, for example, described by Shone et al. (1987) Eur.J. Biochem. 167, pp. 175-180; and by Blaustein et al. (1987) FEBS 226(1), pp. 115-120.

The Shone et al. method employs artificial liposomes loaded withpotassium phosphate buffer (pH 7.2) and radiolabelled NAD. Release of K+and NAD from the liposomes correlates with a positive result for channelforming activity and hence translocation activity. In this regard, K+release from liposomes may be measured using an electrode and NADrelease calculated by measuring the radioactivity in the supernatant(see page 176, column 1, line 33-column 2, line 17).

The Blaustein et al. method employs planar phospholipid bilayermembranes, which are used to test for channel forming activity. In moredetail, salt solutions on either side of the membrane are buffered at adifferent pH—on the cis side, pH 4.7 or 5.5 and on the trans side, pH7.4. The “conjugate” to be tested is added to the cis side of themembrane and electrical measurements are made under voltage clampconditions, in to order to monitor the flow of current across themembrane (see paragraph 2.2, pages 116-118). The presence of an activetranslocation function is confirmed by a steady rate of channel turn-on(i.e. a positive result for channel formation)—see paragraph 3, page118.

Targeting Moiety (TM) function after conjugation may be tested byassaying for the agonist function inherent to the TM. Suitable methodsinclude those described in

Example 1

The ability of the conjugate of the invention to inhibit substance Prelease from nociceptive afferent cells can be assessed using themethods described in Example 15.

In Example 15, a nociceptin-LHN/A conjugate according to the firstaspect of the invention is assessed for its ability to inhibit therelease of substance P from primary nociceptive sensory afferentneurons. As can be seen from Table 1, incubation of the conjugate withcultures of nociceptive afferent neurons results in a significantinhibition of release of substance P (when compared to incubation of thecells with LHN/A alone). The experiment therefore confirms that theconjugate is inhibiting substance P release from these cells.

In use of the present invention, a pain-sensing target cell is selectedin which it is desired to reduce or inhibit the process of exocyticfusion, which exocytic process contributes to the symptoms associatedwith the sensation of pain. For example, the target cell in question maydemonstrate an undesirable phenotype (e.g. an undesirable secretion, orthe expression of an undesirable concentration of membrane receptor,transporter or membrane channel), which contributes to the symptomsassociated with pain. Alternatively, a target cell may be selected inwhich the process of exocytic fusion contributes to the sensation ofpain.

In preferred embodiments of the invention, the target cell is anociceptive sensory afferent cell, preferably a primary nociceptiveafferent cell (e.g. an A-fibre such as an Aδ-fibre or a C-fibre). Thus,the conjugates of the present invention are capable of inhibitingneurotransmitter or neuromodulator (e.g. glutamate, substance P,calcitonin-gene related peptide (CGRP), and/or neuropeptide Y) releasefrom discrete populations to of nociceptive sensory afferent neurons. Inuse, the conjugates reduce or prevent the transmission of sensoryafferent signals (e.g. neurotransmitters or neuromodulators) fromperipheral to central pain fibres, and therefore have application astherapeutic molecules for the treatment of pain, in particular chronicpain.

It is routine to confirm that a TM binds to a nociceptive sensoryafferent. For example, a simple radioactive displacement experiment maybe employed in which tissue or cells representative of the nociceptivesensory afferent (for example DRGs) are exposed to labelled (e.g.tritiated) ligand in the presence of an excess of unlabelled ligand. Insuch an experiment, the relative proportions of non-specific andspecific binding may be assessed, thereby allowing confirmation that theligand binds to the nociceptive sensory afferent target cell.Optionally, the assay may include one or more binding antagonists, andthe assay may further comprise observing a loss of ligand binding.Examples of this type of experiment can be found in Hulme, E. C. (1990),Receptor-binding studies, a brief outline, pp 303-311, in Receptorbiochemistry, A Practical Approach, Ed. E. C. Hulme, Oxford UniversityPress.

According to a second aspect, the present invention provides anon-cytotoxic conjugate for inhibition or reduction of exocytotic fusionin a nociceptive sensory afferent cell, comprising:

-   -   (i) a Targeting Moiety (TM), wherein said TM is an agonist of a        receptor that is present on said nociceptive sensory afferent        cell, and wherein said receptor undergoes endocytosis to be        incorporated into an endosome within the nociceptive sensory        afferent cell;    -   (ii) a DNA sequence encoding a non-cytotoxic protease or a        fragment thereof, wherein the DNA sequence is expressible in the        nociceptive sensory afferent cell and when so expressed provides        a protease or protease fragment capable of cleaving a protein of        the exocytic fusion apparatus of said nociceptive sensory        afferent cell; and    -   (iii) a Translocation Domain, wherein the Translocation Domain        translocates the DNA sequence encoding the protease or protease        fragment from within the endosome, across the endosomal        membrane, and into the nociceptive sensory afferent cell.

In a preferred embodiment of the invention, the target for the TM isselected from the group consisting of: Mrg receptors such as MrgX1,opiod receptors such as OPRD1 and/or OPRM1, BDKRB1 and/or BDKRB2,Tachykinin receptors such as TACR1, TACR2 and/or TACR3, Kappa receptor(OPRK1) and/or ORL₁ receptor.

In one embodiment, the TM is a molecule that binds (preferably thatspecifically binds) to one or more of the above-mentioned receptors. Forexample, the TM is an “agonist” of one or more of the above-mentionedreceptors. The term “agonist” in this context is defined as above.

In one embodiment, the TM comprises or consists of a BAM peptide.Full-length BAM is a 22 amino acid peptide, abbreviated herein asBAM1-22 (represented by SEQ ID NO: 120). In one embodiment, the BAM TMof the invention comprises or consists of a 15 amino acid fragment offull-length BAM peptide and is referred to herein as BAMS-22(represented by SEQ ID NO: 121). In one embodiment, said BAM peptidesbind (preferably specifically bind) to Mrg receptors such as MrgX1.

In one embodiment, the TM comprises or consists of a β-endorphinpeptide. β-endorphin is a 31 amino acid peptide (represented by SEQ IDNO: 126). In one embodiment, said β-endorphin peptide binds (preferablyspecifically binds) opioid receptors such as OPRD1 and/or OPRM1.

In one embodiment, the TM comprises or consists of a bradykinin peptide.bradykinin is a 9 amino acid peptide (represented by SEQ ID NO: 129). Inone embodiment, said bradykinin peptide binds (preferably specificallybinds) bradykinin target receptors BDKRB1 and/or BDKRB2.

In one embodiment, the TM comprises or consists of a des-Arg⁹-BK ligand(represented by SEQ ID NO: 130). The des-Arg9-Bradykinin ligand differsfrom bradykinin ligand by the removal of an arginine residue from theC-terminus. In one embodiment, said des-Arg⁹-BK ligand binds (preferablyspecifically binds) bradykinin target receptors BDKRB1 and/or BDKRB2.

In one embodiment, the TM comprises or consists of a substance Ppeptide. Full length substance P is an 11 amino acid peptide(represented by SEQ ID NO: 134). In one embodiment, the TM comprises orconsists of a substance P analogue, such as the analogue referred toherein as ‘S6’ (represented by SEQ ID NO: 135). In one embodiment, saidsubstance P peptide, or analogue thereof binds (preferably specificallybinds) to Tachykinin receptors such as TACR1, TACR2 and/or TACR3.

In one embodiment, the TM comprises or consists of a dynorphin peptide.The sequence of dynorphin is represented by SEQ ID NO: 101. In oneembodiment, said dynorphin peptide binds (preferably specifically binds)Kappa receptor (OPRK1).

The invention also encompasses fragments, variants, and derivatives andanalogues of the above-mentioned TMs. These fragments, variants, andderivatives and analogues substantially retain the properties that areascribed to said TM. For example, the fragments, variants, andderivatives may retain the ability to bind to their respectivereceptor(s). By way of example, reference is made to the above-mentionedBAMS-22 fragment of the full length BAM1-22 TM as well as the substanceP analogue S6.

In one embodiment, the TM comprises or consist of an amino acid sequencehaving at least 70%, preferably at least 80% (such as at least 82, 84,85, 86, 88 or 89%), more preferably at least 90% (such as at least 91,92, 93 or 94%), and most preferably at least 95% (such as at least 96,97, 98, 99 or 100%) amino acid sequence acid identity to SEQ ID NO: 2,4, 12, 101, 120, 121, 126, 129, 130, 134 and/or 135.

In one embodiment, the TM comprises or consist of an amino acid sequencehaving at least 70%, preferably at least 80% (such as at least 82, 84,85, 86, 88 or 89%), more preferably at least 90% (such as at least 91,92, 93 or 94%), and most preferably at least 95% (such as at least 96,97, 98, 99 or 100%) amino acid sequence acid identity to SEQ ID NO: 120,121, 126, 129, 130, 134 and/or 135.

In one embodiment, the Targeting Moiety comprises or consists of anamino acid sequence according to SEQ ID NO: 120, 121, 126, 129, 130, 134and/or 135 or a fragment comprising or consisting of at least 16 (suchas at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15) contiguous aminoacid residues thereof, or a variant amino acid sequence of said SEQ IDNO: 120, 121, 126, 129, 130, 134 and/or 135 or said fragment is having amaximum of 6 (such as a maximum of 5, 4, 3, 2 or 1) conservative aminoacid substitutions.

DNA encoding a protein of interest can be transfected into eukaryoticcells through receptor-mediated endocytosis of a protein-DNA conjugate,as confirmed by Cotton et al. (Cotton, M., Wagner, E. and Birnstiel, L.(1993) Receptor-mediated transport of DNA into eukaryotic cells. Methodsin Enzymol. 217, 619-645). Several methods exist for condensing DNA to asuitable size using polycationic ligands. These include: polylysine,various cationic peptides and cationic liposomes. Of these, polylysinewas used in the present study because of its successfully reported usein receptor-mediated transfection studies (Cotton et al., 1993).

The DNA sequence encoding the non-cytotoxic protease component may beexpressed under the control of an operably linked promoter present aspart of the agent (e.g. as part of the protease DNA sequence upstream ofthe coding region). Alternatively, expression of the protease componentin the target cell may rely on a promoter present in the target cell.

The DNA sequence encoding the protease component may integrate into aDNA sequence of the target cell. One or more integration site(s) may beprovided as part of the conjugate (e.g. as part of the protease DNAsequence).

The TM, Translocation Domain and protease components of this secondaspect of the invention are as defined for the first aspect of theinvention. Examples 13 and 14 describe the preparation of conjugatesaccording to the second aspect of the invention.

According to a third aspect, the present invention provides apharmaceutical composition comprising a conjugate according to the firstand/or second aspect of the present invention.

The pharmaceutical composition may further comprise apharmaceutically-acceptable carrier, and/or a suitable diluent and/orexcipient, although the exact form of the composition may be tailored tothe mode of administration. Administration is preferably to a mammal,more preferably to a human.

The components of the composition may, for example, be employed in theform of an aerosol or nebulisable solution for inhalation or a sterilesolution for parenteral administration, intra-articular administrationor intra-cranial administration.

The composition may also be administered by i.v. injection, whichincludes the use of pump systems. Spinal injection (e.g. epidural orintrathecal) or indwelling pumps may also be used.

The dosage ranges for administration of the components of the presentinvention are those to produce the desired therapeutic effect. It willbe appreciated that the dosage range required depends on the precisenature of the components, the route of administration, the nature of theformulation, the age of the patient, the nature, extent or severity ofthe patient's condition, contraindications, if any, and the judgement ofthe attending physician.

Suitable daily dosages (for each component) are in the range 0.0001-1mg/kg, preferably 0.0001-0.5 mg/kg, more preferably 0.002-0.5 mg/kg, andparticularly preferably 0.004-0.5 mg/kg. The unit dosage can vary fromless that 1 microgram to 30 mg, but typically will be in the region of0.01 to 1 mg per dose, which may be administered daily or preferablyless frequently, such as weekly or six monthly.

A particularly preferred dosing regimen is based on 2.5 ng of fusionprotein (e.g. CPNv/A) as the 1× dose. In this regard, preferred dosagesare in the range 1×-100× (i.e. 2.5-250 ng). This dosage range issignificantly lower (i.e. at least 10-fold, typically 100-fold lower)than would be employed with other types of analgesic molecules such asNSAIDS, morphine, and gabapentin. Moreover, the above-mentioneddifference is to considerably magnified when the same comparison is madeon a molar basis—this is because the fusion proteins of the presentinvention have a considerably greater Mw than do conventional ‘small’molecule therapeutics.

Wide variations in the required dosage, however, are to be expecteddepending on the is precise nature of the components, and the differingefficiencies of various routes of administration. For example, oraladministration would be expected to require higher dosages thanadministration by intravenous injection.

Variations in these dosage levels can be adjusted using standardempirical routines for optimisation, as is well understood in the art.

Compositions suitable for injection may be in the form of solutions,suspensions or emulsions, or dry powders which are dissolved orsuspended in a suitable vehicle prior to use.

Fluid unit dosage forms are typically prepared utilising a pyrogen-freesterile vehicle. The active ingredients, depending on the vehicle andconcentration used, can be either dissolved or suspended in the vehicle.

Solutions may be used for all forms of parenteral administration, andare particularly used for intravenous injection. In preparing solutionsthe components can be dissolved in the vehicle, the solution being madeisotonic if necessary by addition of sodium chloride and sterilised byfiltration through a sterile filter using aseptic techniques beforefilling into suitable sterile vials or ampoules and sealing.Alternatively, if solution stability is adequate, the solution in itssealed containers may be sterilised by autoclaving.

Advantageously additives such as buffering, solubilising, stabilising,preservative or bactericidal, suspending or emulsifying agents and/orlocal anaesthetic agents may be dissolved in the vehicle.

Dry powders which are dissolved or suspended in a suitable vehicle priorto use may be prepared by filling pre-sterilised drug substance andother ingredients into a sterile to container using aseptic technique ina sterile area.

Alternatively the components of the composition may be dissolved in anaqueous vehicle, the solution is sterilized by filtration anddistributed into suitable containers using aseptic technique in asterile area. The product is then freeze-dried and the is containers aresealed aseptically.

Parenteral suspensions, suitable for intramuscular, subcutaneous orintradermal injection, are prepared in substantially the same manner,except that the sterile components are suspended in the sterile vehicle,instead of being dissolved and sterilisation cannot be accomplished byfiltration. The components may be isolated in a sterile state oralternatively it may be sterilised after isolation, e.g. by gammairradiation.

Advantageously, a suspending agent for example polyvinylpyrrolidone isincluded in the composition(s) to facilitate uniform distribution of thecomponents.

Compositions suitable for administration via the respiratory tractinclude aerosols, nebulisable solutions or microfine powders forinsufflation. In the latter case, particle size of less than 50 microns,especially less than 10 microns, is preferred. Such compositions may bemade up in a conventional manner and employed in conjunction withconventional administration devices.

The compositions described in this invention can be used in vivo, eitherdirectly or as a pharmaceutically acceptable salt, for the treatment ofconditions involving exocytosis (for example secretion, or the deliveryof proteins such as receptors, transporters, and membrane channels tothe plasma membrane of a cell).

According to a fourth aspect, the present invention provides a DNAconstruct that encodes a conjugate according to the first or secondaspects of the invention.

By expressing the construct in a host cell, conjugates of the inventionmay be prepared.

According to a fifth aspect, the present invention provides a method oftreatment of pain by administration to a patient of a conjugate,composition, or construct according to the first to fourth aspects ofthe invention, or any combination thereof.

In a preferred embodiment, the invention provides a method of treatingchronic pain.

According to a sixth aspect, the present invention provides for the useof a conjugate, composition or construct according to the first tofourth aspects of the invention, for the manufacture of a medicament fortreating pain, preferably chronic pain.

According to a further aspect of the present invention, there isprovided use of a conjugate of the invention, for the manufacture of amedicament for treating, preventing or ameliorating pain. According to arelated aspect, there is provided a method of treating, preventing orameliorating pain in a subject, comprising administering to said patienta therapeutically effective amount of a conjugate or composition of theinvention.

The conjugates and compositions described here may be used to treat apatient suffering from one or more types of chronic pain includingneuropathic pain, inflammatory pain, headache pain, somatic pain,visceral pain, and referred pain.

To “treat,” as used here, means to deal with medically. It includes, forexample, administering a compound of the invention to prevent pain or tolessen its severity.

The term “pain,” as used here, means any unpleasant sensory experience,usually associated with a physical disorder. The physical disorder mayor may not be apparent to a clinician. Pain is of two types: chronic andacute. An “acute pain” is a pain of short duration having a suddenonset. One type of acute pain, for example, is cutaneous pain felt oninjury to the skin or other superficial tissues, such as caused by a cutor a burn. Cutaneous nociceptors terminate just below the skin, and dueto the high concentration of nerve endings, produce a well-defined,localized pain of short duration. “Chronic pain” is a pain other than anacute pain. Chronic pain includes neuropathic pain, inflammatory pain,headache pain, somatic pain visceral pain and referred pain.

I. Neuropathic Pain

The compounds of the invention may be used to treat pain caused by orotherwise associated with any of the following neuropathic painconditions. “Neuropathic pain” means abnormal sensory input, resultingin discomfort, from the peripheral nervous system, central nervoussystems, or both.

A. Symptoms of Neuropathic Pain

Symptoms of neuropathic pain can involve persistent, spontaneous pain,as well as allodynia (a painful response to a stimulus that normally isnot painful), hyperalgesia (an accentuated response to a painfulstimulus that usually causes only a mild discomfort, such as a pinprick), or hyperpathia (where a short discomfort becomes a prolongedsevere pain).

B. Causes of Neuropathic Pain

Neuropathic pain may be caused by any of the following.

1. A traumatic insult, such as, for example, a nerve compression injury(e.g., a nerve crush, a nerve stretch, a nerve entrapment or anincomplete nerve transsection); a spinal cord injury (e.g., ahemisection of the spinal cord); a limb amputation; a contusion; aninflammation (e.g., an inflammation of the spinal cord); or a surgicalprocedure.2. An ischemic event, including, for example, a stroke and heart attack.3. An infectious agent4. Exposure to a toxic agent, including, for example, a drug, analcohol, a heavy metal (e.g., lead, arsenic, mercury), an industrialagent (e.g., a solvent, fumes from a glue) or nitrous oxide.5. A disease, including, for example, an inflammatory disorder, aneoplastic tumor, an acquired immune deficiency syndrome (AIDS), Lymesdisease, a leprosy, a metabolic disease, a peripheral nerve disorder,like neuroma, a mononeuropathy or a polyneuropathy.

C. Types of Neuropathic Pain 1. Neuralgia.

A neuralgia is a pain that radiates along the course of one or morespecific nerves usually without any demonstrable pathological change inthe nerve structure. The causes of neuralgia are varied. Chemicalirritation, inflammation, trauma (including surgery), compression bynearby structures (for instance, tumors), and infections may all lead toneuralgia. In many cases, however, the cause is unknown orunidentifiable. Neuralgia is most common in elderly persons, but it mayoccur at any age. A neuralgia, includes, without limitation, atrigeminal neuralgia, a post-herpetic neuralgia, a postherpeticneuralgia, a glossopharyngeal neuralgia, a sciatica and an atypicalfacial pain.

Neuralgia is pain in the distribution of a nerve or nerves. Examples aretrigeminal neuralgia, atypical facial pain, and postherpetic neuralgia(caused by shingles or herpes). The affected nerves are responsible forsensing touch, temperature and pressure in the facial area from the jawto the forehead. The disorder generally causes short episodes ofexcruciating pain, usually for less than two minutes and on only oneside of the face. The pain can be described in a variety of ways such as“stabbing,” “sharp,” “like lightning,” “burning,” and even “itchy”. Inthe atypical form of TN, the pain can also present as severe or merelyaching and last for extended periods. The pain associated with TN isrecognized as one the most excruciating pains that can be experienced.

Simple stimuli such as eating, talking, washing the face, or any lighttouch or sensation can trigger an attack (even the sensation of a gentlebreeze). The attacks can occur in clusters or as an isolated attack.

Symptoms include sharp, stabbing pain or constant, burning pain locatedanywhere, usually on or near the surface of the body, in the samelocation for each episode; pain along the path of a specific nerve;impaired function of affected body part due to pain, or muscle weaknessdue to concomitant motor nerve damage; increased sensitivity of the skinor numbness of the affected skin area (feeling similar to a localanesthetic such as to a Novocaine shot); and any touch or pressure isinterpreted as pain. Movement may also be painful.

Trigeminal neuralgia is the most common form of neuralgia. It affectsthe main sensory nerve of the face, the trigeminal nerve (“trigeminal”literally means “three origins”, is referring to the division of thenerve into 3 branches). This condition involves sudden and short attacksof severe pain on the side of the face, along the area supplied by thetrigeminal nerve on that side. The pain attacks may be severe enough tocause a facial grimace, which is classically referred to as a painfultic (tic douloureux). Sometimes, the cause of trigeminal neuralgia is ablood vessel or small tumor pressing on the nerve. Disorders such asmultiple sclerosis (an inflammatory disease affecting the brain andspinal cord), certain forms of arthritis, and diabetes (high bloodsugar) may also cause trigeminal neuralgia, but a cause is not alwaysidentified. In this condition, certain movements such as chewing,talking, swallowing, or touching an area of the face may trigger a spasmof excruciating pain.

A related but rather uncommon neuralgia affects the glosso-pharyngealnerve, which provides sensation to the throat. Symptoms of thisneuralgia are short, shock-like episodes of pain located in the throat.

Neuralgia may occur after infections such as shingles, which is causedby the varicella-zoster virus, a type of herpesvirus. This neuralgiaproduces a constant burning pain after the shingles rash has healed. Thepain is worsened by movement of or contact with the affected area. Notall of those diagnosed with shingles go on to experience postherpeticneuralgia, which can be more painful than shingles. The pain andsensitivity can last for months or even years. The pain is usually inthe form of an intolerable sensitivity to any touch but especially lighttouch. Postherpetic neuralgia is not restricted to the face; it canoccur anywhere on the body but usually occurs at the location of theshingles rash. Depression is not uncommon due to the pain and socialisolation during the illness.

Postherpetic neuralgia may be debilitating long after signs of theoriginal herpes infection have disappeared. Other infectious diseasesthat may cause neuralgia are syphilis and Lyme disease.

Diabetes is another common cause of neuralgia. This very common medicalproblem affects almost 1 out of every 20 Americans during adulthood.Diabetes damages the tiny arteries that supply circulation to thenerves, resulting in nerve fiber malfunction and sometimes nerve loss.Diabetes can produce almost any neuralgia, including trigeminalneuralgia, carpal tunnel syndrome (pain and numbness of the hand andwrist), and meralgia paresthetica (numbness and pain in the thigh due todamage to the lateral femoral cutaneous nerve). Strict control of bloodsugar may prevent diabetic nerve damage and may accelerate recovery inpatients who do develop neuralgia.

Other medical conditions that may be associated with neuralgias arechronic renal insufficiency and porphyria—a hereditary disease in whichthe body cannot rid itself of certain substances produced after thenormal breakdown of blood in the body. Certain drugs may also cause thisproblem.

2. Deafferentation.

Deafferentation indicates a loss of the sensory input from a portion ofthe body, and can be caused by interruption of either peripheral sensoryfibres or nerves from the central nervous system. A deafferentation painsyndrome, includes, without limitation, an injury to the brain or spinalcord, a post-stroke pain, a phantom pain, a paraplegia, a brachialplexus avulsion injuries, lumbar radiculopathies.

3. Complex Regional Pain Syndromes (CRPSs)

CRPS is a chronic pain syndrome resulting fromsympathetically-maintained pain, and presents in two forms. CRPS 1currently replaces the term “reflex sympathetic dystrophy syndrome”. Itis a chronic nerve disorder that occurs most often in the arms or legsafter a minor or major injury. CRPS 1 is associated with severe pain;changes in the nails, bone, and skin; and an increased sensitivity totouch in the affected limb. CRPS 2 replaces the term causalgia, andresults from an identified injury to the nerve. A CRPS, includes,without limitation, a CRPS Type I (reflex sympathetic dystrophy) and aCRPS Type II (causalgia).

to 4. Neuropathy.

A neuropathy is a functional or pathological change in a nerve and ischaracterized clinically by sensory or motor neuron abnormalities.

Central neuropathy is a functional or pathological change in the centralnervous system.

Peripheral neuropathy is a functional or pathological change in one ormore peripheral nerves. The peripheral nerves relay information fromyour central nervous system (brain and spinal cord) to muscles and otherorgans and from your skin, joints, and other organs back to your brain.Peripheral neuropathy occurs when these nerves fail to carry informationto and from the brain and spinal cord, resulting in pain, loss ofsensation, or inability to control muscles. In some cases, the failureof nerves that control blood vessels, intestines, and other organsresults in abnormal blood pressure, digestion problems, and loss ofother basic body processes. Risk factors for neuropathy includediabetes, heavy alcohol use, and exposure to certain chemicals anddrugs. Some people have a hereditary predisposition for neuropathy.Prolonged pressure on a nerve is another risk for developing a nerveinjury. Pressure injury may be caused by prolonged immobility (such as along surgical procedure or lengthy illness) or compression of a nerve bycasts, splints, braces, crutches, or other devices. Polyneuropathyimplies a widespread process that usually affects both sides of the bodyequally. The symptoms depend on which type of nerve is affected. Thethree main types of nerves are sensory, motor, and autonomic. Neuropathycan affect any one or a combination of all three types of nerves.Symptoms also depend on whether the condition affects the whole body orjust one nerve (as from an injury). The cause of chronic inflammatorypolyneuropathy is an abnormal immune response. The specific antigens,immune processes, and triggering factors are variable and in many casesare unknown. It may occur in association with other conditions such asHIV, inflammatory bowel disease, lupus erythematosis, chronic activehepatitis, and blood cell abnormalities.

Peripheral neuropathy may involve a function or pathological change to asingle nerve or nerve group (monneuropathy) or a function orpathological change affecting multiple nerves (polyneuropathy).

Peripheral Neuropathies Hereditary Disorders

-   -   Charcot-Marie-Tooth disease    -   Friedreich's ataxia

Systemic or Metabolic Disorders

-   -   Diabetes (diabetic neuropathy)    -   Dietary deficiencies (especially vitamin B-12)    -   Excessive alcohol use (alcoholic neuropathy)    -   Uremia (from kidney failure)    -   Cancer

Infectious or Inflammatory Conditions

-   -   AIDS    -   Hepatitis    -   Colorado tick fever    -   diphtheria    -   Guillain-Barre syndrome    -   HIV infection without development of AIDS    -   leprosy    -   Lyme    -   polyarteritis nodosa    -   rheumatoid arthritis    -   sarcoidosis    -   Sjogren syndrome    -   syphilis    -   systemic lupus erythematosus    -   amyloid

Exposure to Toxic Compounds

-   -   sniffing glue or other toxic compounds    -   nitrous oxide    -   industrial agents—especially solvents    -   heavy metals (lead, arsenic, mercury, etc.)    -   Neuropathy secondary to drugs like analgesic nephropathy

Miscellaneous Causes

-   -   ischemia (decreased oxygen/decreased blood flow)    -   prolonged exposure to cold temperature

a. Polyneuropathy

Polyneuropathy is a peripheral neuropathy involving the loss of movementor is sensation to an area caused by damage or destruction to multipleperipheral nerves. Polyneuropathic pain, includes, without limitation,post-polio syndrome, postmastectomy syndrome, diabetic neuropathy,alcohol neuropathy, amyloid, toxins, AIDS, hypothyroidism, uremia,vitamin deficiencies, chemotherapy-induced pain, 2′,3′-didexoycytidine(ddC) treatment, Guillain-Barré syndrome or Fabry's disease.

b. Mononeuropathy

Mononeuropathy is a peripheral neuropathy involving loss of movement orsensation to an area caused by damage or destruction to a singleperipheral nerve or nerve group. Mononeuropathy is most often caused bydamage to a local area resulting from injury or trauma, althoughoccasionally systemic disorders may cause isolated nerve damage (as withmononeuritis multiplex). The usual causes are direct trauma, prolongedpressure on the nerve, and compression of the nerve by swelling orinjury to nearby body structures. The damage includes destruction of themyelin sheath (covering) of the nerve or of part of the nerve cell (theaxon). This damage slows or prevents conduction of impulses through thenerve. Mononeuropathy may involve any part of the body. Mononeuropathicpain, includes, without limitation, a sciatic nerve dysfunction, acommon peroneal nerve dysfunction. a radial nerve dysfunction, an ulnarnerve dysfunction, a cranial mononeuropathy VI, a cranial mononeuropathyVII, a cranial mononeuropathy III (compression type), a cranialmononeuropathy III (diabetic type), an axillary nerve dysfunction, acarpal tunnel syndrome, a femoral nerve dysfunction, a tibial nervedysfunction, a Bell's palsy, a thoracic outlet syndrome, a carpal tunnelsyndrome and a sixth (abducent) nerve palsy

c. Generalized Peripheral Neuropathies

Generalized peripheral neuropathies are symmetrical, and usually due tovarious systematic illnesses and disease processes that affect theperipheral nervous system in its entirety. They are further subdividedinto several categories:

i. Distal axonopathies are the result of some metabolic or toxicderangement of neurons. They may be caused by metabolic diseases such asdiabetes, renal failure, deficiency syndromes such as malnutrition andalcoholism, or the effects of toxins or drugs. Distal axonopathy (akadying back neuropathy) is a type of peripheral neuropathy that resultsfrom some metabolic or toxic derangement of peripheral nervous system(PNS) neurons. It is the most common response of nerves to metabolic ortoxic disturbances, and as such may be caused by metabolic diseases suchas diabetes, is renal failure, deficiency syndromes such as malnutritionand alcoholism, or the effects of toxins or drugs. The most common causeof distal axonopathy is diabetes, and the most common distal axonopathyis diabetic neuropathy.

ii. Myelinopathies are due to a primary attack on myelin causing anacute failure of impulse conduction. The most common cause is acuteinflammatory demyelinating polyneuropathy (AIDP; aka Guillain-Barrésyndrome), though other causes include chronic inflammatorydemyelinating syndrome (CIDP), genetic metabolic disorders (e.g.,leukodystrophy), or toxins. Myelinopathy is due to primary destructionof myelin or the myelinating Schwann cells, which leaves the axonintact, but causes an acute failure of impulse conduction. Thisdemyelination slows down or completely blocks the conduction ofelectrical impulses through the nerve. The most common cause is acuteinflammatory demyelinating polyneuropathy (AIDP, better known asGuillain-Barré syndrome), though other causes include chronicinflammatory demyelinating polyneuropathy (CIDP), genetic metabolicdisorders (e.g., leukodystrophy or Charcot-Marie-Tooth disease), ortoxins.

iii. Neuropathies are the result of destruction of peripheral nervoussystem (PNS) neurons. They may be caused by motor neurone diseases,sensory neuronopathies (e.g., Herpes zoster), toxins or autonomicdysfunction. Neurotoxins may cause neuronopathies, such as thechemotherapy agent vincristine. Neuropathy is dysfunction due to damageto neurons of the peripheral nervous system (PNS), resulting in aperipheral neuropathy. It may be caused by motor neurone diseases,sensory neuronopathies (e.g., Herpes zoster), toxic substances orautonomic dysfunction. A person with neuropathy may present in differentways, depending on the cause, the way it affects the nerve cells, andthe type of nerve cell that is most affected.

iv. Focal Entrapment Neuropathies (e.g., Carpal Tunnel Syndrome).

II. Inflammatory Pain

The compounds of the invention may be used to treat pain caused by orotherwise associated with any of the following inflammatory conditions

A. Arthritic Disorder

Arthritic disorders include, for example, a rheumatoid arthritis; ajuvenile rheumatoid is arthritis; a systemic lupus erythematosus (SLE);a gouty arthritis; a scleroderma; an osteoarthritis; a psoriaticarthritis; an ankylosing spondylitis; a Reiter's syndrome (reactivearthritis); an adult Still's disease; an arthritis from a viralinfection; an arthritis from a bacterial infection, such as, e.g., agonococcal arthritis and a non-gonococcal bacterial arthritis (septicarthritis); a Tertiary Lyme disease; a tuberculous arthritis; and anarthritis from a fungal infection, such as, e.g. a blastomycosis

B. Autoimmune Diseases

Autoimmune diseases include, for example, a Guillain-Barré syndrome, aHashimoto's thyroiditis, a pernicious anemia, an Addison's disease, atype I diabetes, a systemic lupus erythematosus, a dermatomyositis, aSjogren's syndrome, a lupus erythematosus, a multiple sclerosis, amyasthenia gravis, a Reiter's syndrome and a Grave's disease.

C. Connective Tissue Disorder

Connective tissue disorders include, for example, a spondyloarthritis adermatomyositis, and a fibromyalgia.

D. Injury

Inflammation caused by injury, including, for example, a crush,puncture, stretch of a tissue or joint, may cause chronic inflammatorypain.

E. Infection

Inflammation caused by infection, including, for example, a tuberculosisor an interstitial keratitis may cause chronic inflammatory pain.

F. Neuritis

Neuritis is an inflammatory process affecting a nerve or group ofnerves. Symptoms depend on the nerves involved, but may include pain,paresthesias, paresis, or hypesthesia (numbness).

Examples include:

a. Brachial neuritis

b. Retrobulbar neuropathy, an inflammatory process affecting the part ofthe optic nerve lying immediately behind the eyeball.

c. Optic neuropathy, an inflammatory process affecting the optic nervecausing sudden, reduced vision in the affected eye. The cause of opticneuritis is unknown. The sudden inflammation of the optic nerve (thenerve connecting the eye and the brain) leads to swelling anddestruction of the myelin sheath. The inflammation may occasionally bethe result of a viral infection, or it may be caused by autoimmunediseases such as multiple sclerosis. Risk factors are related to thepossible causes.

d. Vestibular neuritis, a viral infection causing an inflammatoryprocess affecting the vestibular nerve.

G. Joint Inflammation

Inflammation of the joint, such as that caused by bursitis ortendonitis, for example, may cause chronic inflammatory pain.

III. Headache Pain

The compounds of the invention may be used to treat pain caused by orotherwise associated with any of the following headache conditions. Aheadache (medically known as cephalgia) is a condition of mild to severepain in the head; sometimes neck or upper back pain may also beinterpreted as a headache. It may indicate an underlying local orsystemic disease or be a disorder in itself.

A. Muscular/Myogenic Headache

Muscular/myogenic headaches appear to involve the tightening or tensingof facial and neck muscles; they may radiate to the forehead. Tensionheadache is the most common form of myogenic headache.

A tension headache is a condition involving pain or discomfort in thehead, scalp, or neck, usually associated with muscle tightness in theseareas. Tension headaches result from the contraction of neck and scalpmuscles. One cause of this muscle contraction is a response to stress,depression or anxiety. Any activity that causes the head to be held inone position for a long time without moving can cause a headache. Suchactivities include typing or use of computers, fine work with the hands,and use of a microscope. Sleeping in a cold room or sleeping with theneck in an abnormal position may also trigger this type of headache. Atension-type headache, includes, without limitation, an episodic tensionheadache and a chronic tension headache.

B. Vascular Headache

The most common type of vascular headache is migraine. Other kinds ofvascular headaches include cluster headaches, which cause repeatedepisodes of intense pain, and headaches resulting from high bloodpressure

1. Migraine

A migraine is a heterogeneous disorder that generally involves recurringheadaches. Migraines are different from other headaches because theyoccur with other symptoms, such as, e.g., nausea, vomiting, orsensitivity to light. In most people, a throbbing pain is felt only onone side of the head. Clinical features such as type of aura symptoms,presence of prodromes, or associated symptoms such as vertigo, may beseen in subgroups of patients with different underlyingpathophysiological and genetic mechanisms. A migraine headache,includes, without limitation, a migraine without aura (common migraine),a migraine with aura (classic migraine), a menstrual migraine, amigraine equivalent (acephalic headache), a complicated migraine, anabdominal migraine and a mixed tension migraine.

2. Cluster Headache

Cluster headaches affect one side of the head (unilateral) and may beassociated with tearing of the eyes and nasal congestion. They occurs inclusters, happening repeatedly every day at the same time for severalweeks and then remitting.

D. High Blood Pressure Headache E. Traction and Inflammatory Headache

Traction and inflammatory headaches are usually symptoms of otherdisorders, ranging from stroke to sinus infection.

F. Hormone Headache G. Rebound Headache

Rebound headaches, also known as medication overuse headaches, occurwhen is medication is taken too frequently to relieve headache. Reboundheadaches frequently occur daily and can be very painful.

H. Chronic Sinusitis Headache

Sinusitis is inflammation, either bacterial, fungal, viral, allergic orautoimmune, of the paranasal sinuses. Chronic sinusitis is one of themost common complications of the common cold. Symptoms include: Nasalcongestion; facial pain; headache; fever; general malaise; thick greenor yellow discharge; feeling of facial ‘fullness’ worsening on bendingover. In a small number of cases, chronic maxillary sinusitis can alsobe brought on by the spreading of bacteria from a dental infection.Chronic hyperplastic eosinophilic sinusitis is a noninfective form ofchronic sinusitis.

I. An Organic Headache J. Ictal Headaches

Ital headaches are headaches associated with seizure activity.

IV. Somatic Pain

The compounds of the invention may be used to treat pain caused by orotherwise associated with any of the following somatic pain conditions.Somatic pain originates from ligaments, tendons, bones, blood vessels,and even nerves themselves. It is detected with somatic nociceptors. Thescarcity of pain receptors in these areas produces a dull,poorly-localized pain of longer duration than cutaneous pain; examplesinclude sprains and broken bones. Additional examples include thefollowing.

A. Excessive Muscle Tension

Excessive muscle tension can be caused, for example, by a sprain or astrain.

B. Repetitive Motion Disorders

Repetitive motion disorders can result from overuse of the hands,wrists, elbows, shoulders, neck, back, hips, knees, feet, legs, orankles.

C. Muscle Disorders

Muscle disorders causing somatic pain include, for example, apolymyositis, a dermatomyositis, a lupus, a fibromyalgia, a polymyalgiarheumatica, and a rhabdomyolysis.

D. Myalgia

Myalgia is muscle pain and is a symptom of many diseases and disorders.The most common cause for myalgia is either overuse or over-stretchingof a muscle or group of muscles. Myalgia without a traumatic history isoften due to viral infections. Longer-term myalgias may be indicative ofa metabolic myopathy, some nutritional deficiencies or chronic fatiguesyndrome.

E. Infection

Infection can cause somatic pain. Examples of such infection include,for example, an abscess in the muscle, a trichinosis, an influenza, aLyme disease, a malaria, a Rocky Mountain spotted fever, Avianinfluenza, the common cold, community-acquired pneumonia, meningitis,monkeypox, Severe Acute Respiratory Syndrome, toxic shock syndrome,trichinosis, typhoid fever, and upper respiratory tract infection.

F. Drugs

Drugs can cause somatic pain. Such drugs include, for example, cocaine,a statin for lowering cholesterol (such as atorvastatin, simvastatin,and lovastatin), and an ACE inhibitor for lowering blood pressure (suchas enalapril and captopril)

V. Visceral Pain

The compounds of the invention may be used to treat pain caused by orotherwise associated with any of the following visceral pain conditions.Visceral pain originates from body's viscera, or organs. Visceralnociceptors are located within body organs and internal cavities. Theeven greater scarcity of nociceptors in these areas produces pain thatis usually more aching and of a longer duration than somatic pain.Visceral pain is extremely difficult to localise, and several injuriesto visceral tissue exhibit “referred” pain, where the sensation islocalised to an area completely unrelated to the site of injury.Examples of visceral pain include the following.

A. Functional Visceral Pain

Functional visceral pain includes, for example, an irritable bowelsyndrome and a chronic functional abdominal pain (CFAP), a functionalconstipation and a functional dyspepsia, a non-cardiac chest pain (NCCP)and a chronic abdominal pain.

B. Chronic Gastrointestinal Inflammation

Chronic gastrointestinal inflammation includes, for example, agastritis, an inflammatory bowel disease, like, e.g., a Crohn's disease,an ulcerative colitis, a microscopic colitis, a diverticulitis and agastroenteritis; an interstitial cystitis; an intestinal ischemia; acholecystitis; an appendicitis; a gastroesophageal reflux; an ulcer, anephrolithiasis, an urinary tract infection, a pancreatitis and ahernia.

C. Autoimmune Pain

Autoimmune pain includes, for example, a sarcoidosis and a vasculitis.

D. Organic Visceral Pain

Organic visceral pain includes, for example, pain resulting from atraumatic, inflammatory or degenerative lesion of the gut or produced bya tumor impinging on sensory innervation.

E. Treatment-Induced Visceral Pain

Treatment-induced visceral pain includes, for example, a pain attendantto chemotherapy therapy or a pain attendant to radiation therapy.

VI. Referred Pain

The compounds of the invention may be used to treat pain caused by orotherwise associated with any of the following referred pain conditions.

Referred pain arises from pain localized to an area separate from thesite of pain stimulation. Often, referred pain arises when a nerve iscompressed or damaged at or is near its origin. In this circumstance,the sensation of pain will generally be felt in the territory that thenerve serves, even though the damage originates elsewhere. A commonexample occurs in intervertebral disc herniation, in which a nerve rootarising from the spinal cord is compressed by adjacent disc material.Although pain may arise from the damaged disc itself, pain will also befelt in the region served by the compressed nerve (for example, thethigh, knee, or foot). Relieving the pressure on the nerve root mayameliorate the referred pain, provided that permanent nerve damage hasnot occurred. Myocardial ischaemia (the loss of blood flow to a part ofthe heart muscle tissue) is possibly the best known example of referredpain; the sensation can occur in the upper chest as a restrictedfeeling, or as an ache in the left shoulder, arm or even hand.

DEFINITIONS SECTION

Exocytic fusion is a process by which intracellular molecules aretransported from the cytosol of a pain-sensing target cell to the plasma(i.e. cell) membrane thereof. Thereafter, the intracellular moleculesmay become displayed on the outer surface of the plasma membrane, or maybe secreted into the extracellular environment.

In a healthy individual, the rate of exocytic fusion is carefullyregulated and allows control of the transport of molecules between thecytosol and the plasma membrane of a pain-sensing cell. For example,regulation of the exocytic cycle allows control of the density ofreceptors, transporters, or membrane channels present at the cell'ssurface, and/or allows control of the secretion rate of intracellularcomponents (e.g. neurotransmitters) from the cytosol of the cell.

However, in an unhealthy individual, the regulation of exocytic fusionmay be modified. For example, exocytic fusion may cause affectedpain-sensing cells to enter a state of hypersecretion. Alternatively,exocytic fusion may result in the display of an increased concentrationof receptors, transporters, or membrane channels present on the surfaceof the pain-sensing, which may expose the cell to undesirable externalstimuli. Thus, the process of exocytic fusion may contribute to theprogression and/or severity of pain, and therefore provides a target fortherapeutic intervention.

It should also be appreciated that otherwise normal rates of cellularexocytic fusion may contribute to the progression and severity of painin compromised patients. Thus, by targeting exocytic fusion inaccordance with the present invention, it is also possible to providetherapy in such patients

Targeting Moiety (TM) means any chemical structure associated with aconjugate that functionally interacts with a receptor, e.g. an ORL₁receptor, to cause a physical association between the conjugate and thesurface of a pain-sensing target cell. The term TM embraces any molecule(i.e. a naturally occurring molecule, or a chemically/physicallymodified variant thereof) that is capable of binding to a receptor onthe target cell, which receptor is capable of internalisation (e.g.endosome formation)—also referred to as receptor-mediated endocytosis.The TM may possess an endosomal membrane translocation domain, in whichcase separate TM and Translocation Domain components need not be presentin an agent of the present invention. The TM of the present inventionbinds (preferably specifically binds) to a nociceptive sensory afferent(e.g. a primary nociceptive afferent). In this regard, specificallybinds means that the TM binds to a nociceptive sensory afferent (e.g. aprimary nociceptive afferent) with a greater affinity than it binds toother neurons such as non-nociceptive afferents, and/or to motor neurons(i.e. the natural target for clostridial neurotoxin holotoxin). The term“specifically binding” can also mean that a given TM binds to a givenreceptor, for Mrg receptors such as MrgX1, opiod receptors such as OPRD1and/or OPRM1, BDKRB1 and/or BDKRB2, Tachykinin receptors such as TACR1,TACR2 and/or TACR3, Kappa receptor (OPRK1) and/or ORL₁ receptor, with abinding affinity (Ka) of 10⁶ M⁻¹ or greater, preferably 10⁷ M⁻¹ orgreater, more preferably 10⁸ M⁻¹ or greater, and most preferably, 10⁹M⁻¹ or greater.

The term “fragment” means a peptide having at least thirty-five,preferably at least twenty-five, more preferably at least twenty, andmost preferably at least 19, 18, 17, 16, 15, 14, 13, 12, 11, 10 9, 8, 7,6 or 5 amino acid residues of the protein (e.g. TM) in question. In oneembodiment, the first amino acid residue of the fragment is theN-terminal amino acid residue of the TM from which the fragment has beenderived.

An example of a “variant” is a peptide or peptide fragment of a TM thatcontains one or is more analogues of an amino acid (e.g. an unnaturalamino acid), or a substituted linkage.

A “derivative” comprises the TM in question, and a further peptidesequence. The further peptide sequence should preferably not interferewith the basic folding and thus conformational structure of the TM. Twoor more peptides (or fragments, or variants) may be joined together toform a derivative. Alternatively, a peptide (or fragment, or variant)may be joined to an unrelated molecule (e.g. a second, unrelatedpeptide). Derivatives may be chemically synthesized, but will betypically prepared by recombinant nucleic acid methods. Additionalcomponents such as lipid, and/or polysaccharide, and/or polyketidecomponents may be included.

The term non-cytotoxic means that the protease molecule in question doesnot kill the pain-sensing target cell to which it has been re-targeted.

The “protease cleavage site” of the present invention allows cleavage(preferably controlled cleavage) of the conjugate at a position betweenthe non-cytotoxic protease component and the TM component. In oneembodiment, the conjugate may include more than one proteolytic cleavagesite. However, where two or more such sites exist, they are different,thereby substantially preventing the occurrence of multiple cleavageevents in the presence of a single protease. In another embodiment, itis preferred that the conjugate has a single protease cleavage site. Theprotease cleavage sequence(s) may be introduced (and/or any inherentcleavage sequence removed) at the DNA level by conventional means, suchas by site-directed mutagenesis. Screening to confirm the presence ofcleavage sequences may be performed manually or with the assistance ofcomputer software (e.g. the MapDraw program by DNASTAR, Inc.).

Whilst any protease cleavage site may be employed, the following arepreferred:

Enterokinase (DDDDK↓) SEQ ID NO: 171 Factor Xa (IEGR↓/IDGR↓)SEQ ID NO: 172 TEV(Tobacco Etch virus) (ENLYFQ↓G) SEQ ID NO: 173Thrombin (LVPR↓GS) SEQ ID NO: 174 PreScission (LEVLFQ↓GP).SEQ ID NO: 175

Also embraced by the term protease cleavage site is an intein, which isa self-cleaving sequence. The self-splicing reaction is controllable,for example by varying the concentration of reducing agent present.

SEQ ID NOs:

Where an initial Met amino acid residue or a corresponding initial codonis indicated in any of the following SEQ ID NOs, said residue/codon isoptional.

-   SEQ ID NO:1 DNA sequence of N[1-17]-   SEQ ID NO:2 Protein Sequence of N[1-17]-   SEQ ID NO:3 DNA sequence of N[1-11]-   SEQ ID NO:4 Protein sequence of N[1-11]-   SEQ ID NO:5 DNA sequence of N[[Y10]1-11]-   SEQ ID NO:6 Protein sequence of N[[Y10]1-11]-   SEQ ID NO:7 DNA sequence of N[[Y11]1-11]-   SEQ ID NO:8 Protein sequence of N[[Y11]1-11]-   SEQ ID NO:9 DNA sequence of N[[Y14]1-17]-   SEQ ID NO:10 Protein sequence of N[[Y14]1-17]-   SEQ ID NO:11 DNA sequence of N[1-13]-   SEQ ID NO:12 Protein sequence of N[1-13]-   SEQ ID NO:13 DNA sequence of Nv (also known as N[[R14K15]1-17])-   SEQ ID NO:14 Protein sequence of Nv (also known as N[[R14K15]1-17])-   SEQ ID NO:15 DNA sequence of N[1-17]-LH_(N)/A fusion protein-   SEQ ID NO:16 Protein sequence of N[1-17]-LH_(N)/A fusion protein-   SEQ ID NO:17 DNA sequence of N[[Y11]1-11]-LHN/A fusion protein-   SEQ ID NO:18 Protein sequence of N[[Y11]1-11]-LHN/A fusion protein-   SEQ ID NO:19 DNA sequence of N[1-13]-LHN/A fusion protein-   SEQ ID NO:20 Protein sequence of N[1-13]-LHN/A fusion protein-   SEQ ID NO:21 DNA sequence of LHN/A-N[1-17] fusion protein-   SEQ ID NO:22 Protein sequence of LHN/A-N[1-17] fusion protein-   SEQ ID NO:23 DNA sequence of LHN/C-N[1-11] fusion protein-   SEQ ID NO:24 Protein sequence of LHN/C-N[1-11] fusion protein-   SEQ ID NO:25 DNA sequence of N[[Y14]1-17]-LHN/C fusion protein-   SEQ ID NO:26 Protein sequence of N[[Y14]1-17]-LHN/C fusion protein-   SEQ ID NO:27 DNA sequence of the LC/A-   SEQ ID NO:28 DNA sequence of the H_(N)/A-   SEQ ID NO:29 DNA sequence of the LC/B-   SEQ ID NO:30 DNA sequence of the H_(N)/B-   SEQ ID NO:31 DNA sequence of the LC/C-   SEQ ID NO:32 DNA sequence of the H_(N)/C-   SEQ ID NO:33 DNA sequence of the CPN-A linker-   SEQ ID NO:34 DNA sequence of the A linker-   SEQ ID NO:35 DNA sequence of the N-terminal presentation nociceptin    insert-   SEQ ID NO:36 DNA sequence of the CPN-C linker-   SEQ ID NO:37 DNA sequence of the CPBE-A linker-   SEQ ID NO:38 DNA sequence of the CPNvar-A linker-   SEQ ID NO:39 DNA sequence of the LC/A-CPN-H_(N)/A fusion-   SEQ ID NO:40 Protein sequence of the LC/A-CPN-H_(N)/A fusion-   SEQ ID NO:41 DNA sequence of the N-LC/A-H_(N)/A fusion-   SEQ ID NO:42 Protein sequence of the N-LC/A-H_(N)/A fusion-   SEQ ID NO:43 DNA sequence of the LC/C-CPN-H_(N)/C fusion-   SEQ ID NO:44 Protein sequence of the LC/C-CPN-H_(N)/C fusion-   SEQ ID NO:45 DNA sequence of the LC/C-CPN-H_(N)/C (A-linker) fusion-   SEQ ID NO:46 Protein sequence of the LC/C-CPN-H_(N)/C (A-linker)    fusion-   SEQ ID NO:47 DNA sequence of the LC/A-CPME-H_(N)/A fusion-   SEQ ID NO:48 Protein sequence of the LC/A-CPME-H_(N)/A fusion-   SEQ ID NO:49 DNA sequence of the LC/A-CPBE-H_(N)/A fusion-   SEQ ID NO:50 Protein sequence of the LC/A-CPBE-H_(N)/A fusion-   SEQ ID NO:51 DNA sequence of the LC/A-CPNv-H_(N)/A fusion-   SEQ ID NO:52 Protein sequence of the LC/A-CPNv-H_(N)/A fusion-   SEQ ID NO:53 DNA sequence of the LC/A-CPN[1-11]-HN/A fusion-   SEQ ID NO:54 Protein sequence of the LC/A-CPN[1-11]-HN/A fusion-   SEQ ID NO:55 DNA sequence of the LC/A-CPN[[Y10]1-11]-HN/A fusion-   SEQ ID NO:56 Protein sequence of the LC/A-CPN[[Y10]1-11]-HN/A fusion-   SEQ ID NO:57 DNA sequence of the LC/A-CPN[[Y11]1-11]-HN/A fusion-   SEQ ID NO:58 Protein sequence of the LC/A-CPN[[Y11]1-11]-HN/A fusion-   SEQ ID NO:59 DNA sequence of the LC/A-CPN[[Y14]1-17]-HN/A fusion-   SEQ ID NO:60 Protein sequence of the LC/A-CPN[[Y14]1-17]-HN/A fusion-   SEQ ID NO:61 DNA sequence of the LC/A-CPN[1-13]-HN/A fusion-   SEQ ID NO:62 Protein sequence of the LC/A-CPN[1-13]-HN/A fusion-   SEQ ID NO:63 DNA sequence of the nociceptin-spacer-LC/A-H_(N)/A    fusion-   SEQ ID NO:64 Protein sequence of the nociceptin-spacer-LC/A-H_(N)/A    fusion-   SEQ ID NO:65 DNA sequence of the CPN-A GS10 linker-   SEQ ID NO:66 DNA sequence of the CPN-A GS15 linker-   SEQ ID NO:67 DNA sequence of the CPN-A GS25 linker-   SEQ ID NO:68 DNA sequence of the CPN-A GS30 linker-   SEQ ID NO:69 DNA sequence of the CPN-A HX27 linker-   SEQ ID NO:70 DNA sequence of the LC/A-CPN(GS15)-H_(N)/A fusion-   SEQ ID NO:71 Protein sequence of the LC/A-CPN(GS15)-H_(N)/A fusion-   SEQ ID NO:72 DNA sequence of the LC/A-CPN(GS25)-H_(N)/A fusion-   SEQ ID NO:73 Protein sequence of the LC/A-CPN(GS25)-H_(N)/A fusion-   SEQ ID NO:74 DNA sequence of the CPNvar-A Enterokinase activatable    linker-   SEQ ID NO:75 DNA sequence of the LC/A-CPNv(Ek)-H_(N)/A fusion-   SEQ ID NO:76 Protein sequence of the LC/A-CPNv(Ek)-H_(N)/A fusion-   SEQ ID NO:77 DNA sequence of the CPNvar-A linker-   SEQ ID NO:78 DNA sequence of the LC/C-CPNv-H_(N)/C fusion (act. A)-   SEQ ID NO:79 Protein sequence of the LC/C-CPNv-H_(N)/C fusion (act.    A)-   SEQ ID NO:80 DNA sequence of the LC/A-CPLE-H_(N)/A fusion-   SEQ ID NO:81 Protein sequence of the LC/A-CPLE-H_(N)/A fusion-   SEQ ID NO:82 DNA sequence of the LC/A-CPOP-H_(N)/A fusion-   SEQ ID NO:83 Protein sequence of the LC/A-CPOP-H_(N)/A fusion-   SEQ ID NO:84 DNA sequence of the LC/A-CPOPv-H_(N)/A fusion-   SEQ ID NO:85 Protein sequence of the LC/A-CPOPv-H_(N)/A fusion-   SEQ ID NO:86 DNA sequence of the IgA protease-   SEQ ID NO:87 DNA sequence of the IgA-CPNv-H_(N)/A fusion-   SEQ ID NO:88 Protein sequence of the IgA-CPNv-H_(N)/A fusion-   SEQ ID NO:89 DNA sequence of the FXa-HT-   SEQ ID NO:90 DNA sequence of the CPNv-A-FXa-HT-   SEQ ID NO:91 Protein sequence of the CPNv-A-FXa-HT fusion-   SEQ ID NO:92 DNA sequence of the DT translocation domain-   SEQ ID NO:93 DNA sequence of the CPLE-DT-A-   SEQ ID NO:94 Protein sequence of the CPLE-DT-A fusion-   SEQ ID NO:95 DNA sequence of the TeNT LC-   SEQ ID NO:96 DNA sequence of the CPNv-TENT LC-   SEQ ID NO:97 Protein sequence of the CPNV-TeNT LC fusion-   SEQ ID NO:98 DNA sequence of the CPNvar-C linker-   SEQ ID NO:99 DNA sequence of the LC/C-CPNv-H_(N)/C fusion (act. C)-   SEQ ID NO:100 Protein sequence of the LC/C-CPNv-H_(N)/C fusion (act.    C)-   SEQ ID NO:101 Protein sequence of dynorphin-   SEQ ID NO:102 DNA sequence of LC/A-CPDY-HN/A fusion-   SEQ ID NO:103 Protein sequence of LC/A-CPDY-HN/A fusion-   SEQ ID NO:104 Protein sequence of LC/A-CPDY(GS10)-H_(N)/A fusion-   SEQ ID NO:105 Protein sequence of LC/A-CPDY(GS15)-H_(N)/A fusion-   SEQ ID NO:106 Protein sequence of LC/A-CPDY(GS25)-H_(N)/A fusion-   SEQ ID NO:107 Protein sequence of LC/C-CPDY-HN/C fusion-   SEQ ID NO:108 Protein sequence of IgA-CPDY-HN/A fusion-   SEQ ID NO:109 Protein sequence of CPDY-TeNT LC fusion-   SEQ ID NO:110 Protein sequence of LC/A-CPDY-H_(N)/A (GS30) fusion-   SEQ ID NO:111 Protein sequence of LC/A-CPDY-H_(N)/A (HX27) fusion-   SEQ ID NO:112 Protein sequence of LC/B-CPDY-H_(N)/B fusion-   SEQ ID NO:113 Protein sequence of LC/A-CPDY1-13-H_(N)/A fusion-   SEQ ID NO:114 Protein sequence of LC/A-CPDY(D15A)-H_(N)/A fusion-   SEQ ID NO:115 Protein sequence of LC/A-CPDY(D15A)-H_(N)/A (GS30)    fusion-   SEQ ID NO:116 Protein sequence of LC/A-CPDY1-13-H_(N)/A (GS30)    fusion-   SEQ ID NO:117 Protein sequence of LC/A-CPDY(I8RP10RD15A)-H_(N)/A    fusion-   SEQ ID NO:118 Protein sequence of LC/A-CPDY(I8RP10R)1-13-H_(N)/A    fusion-   SEQ ID NO:119 Protein sequence of LC/A-CPDNv9-H_(N)/A fusion-   SEQ ID NO:120 Protein sequence of BAM1-22-   SEQ ID NO:121 Protein sequence of BAMS-22-   SEQ ID NO:122 DNA sequence of LC/A-CPBAM(1-22)-H_(N)/A fusion-   SEQ ID NO:123 Protein sequence of LC/A-CPBAM(1-22)-H_(N)/A fusion-   SEQ ID NO:124 Protein sequence of LC/A-H_(N)/A-BAM(8-22)-H_(N)/A    fusion-   SEQ ID NO:125 Protein sequence of LC/A-CPBAM(8-22)-H_(N)/A fusion-   SEQ ID NO:126 Protein sequence of β-endorphin-   SEQ ID NO:127 Protein sequence of LC/D-CPBE-H_(N)/D fusion-   SEQ ID NO:128 Protein sequence of LC/B-CPBE-H_(N)/B fusion-   SEQ ID NO:129 Protein sequence of bradykinin-   SEQ ID NO:130 Protein sequence of des Arg⁹-BK-   SEQ ID NO:131 DNA sequence of LC/A-H_(N)/A-BK fusion-   SEQ ID NO:132 Protein sequence of LC/A-H_(N)/A-BK fusion-   SEQ ID NO:133 Protein sequence of LC/A-H_(N)/A-des Arg⁹-BK fusion-   SEQ ID NO:134 Protein sequence of Substance P-   SEQ ID NO:135 Protein sequence of Substance P analogue (S60)-   SEQ ID NO:136 Protein sequence of LC/A-HN/A-S6 fusion-   SEQ ID NO:137 Protein sequence of LC/B-CPNv-H_(N)/B fusion-   SEQ ID NO:138 Protein sequence of LC/D-CPNv-H_(N)D fusion[-   SEQ ID NO:139 DNA sequence of LC/D-   SEQ ID NO:140 DNA sequence of H_(N)/D-   SEQ ID NO:141 Protein sequence of LHA-EN-CPDNv9-   SEQ ID NO:142 Protein sequence of LHA-CPOPv-   SEQ ID NO:143 Protein sequence of LHA-EN-CPNv-   SEQ ID NO:144 Protein sequence of LHA-Xa-GS-BA-ss-   SEQ ID NO:145 Protein sequence of LHA-EK-CPBAM8-22-GS20-HnA-HT-   SEQ ID NO:146 Protein sequence of LHA-EK-CPBAM1-22-GS20-HnA-HT-   SEQ ID NO:147 Protein sequence of LHA-Xa-CPBE-HT-   SEQ ID NO:148 Protein sequence of LHA-Xa-CPBE-HT-   SEQ ID NO:149 Protein sequence of LHB-Xa-CPBE-HT-   SEQ ID NO:150 Protein sequence of LHD-Xa-CPBE-HT-   SEQ ID NO:151 Protein sequence of LHA-BK-   SEQ ID NO:152 Protein sequence of LHA-EN-CPDY-HT-   SEQ ID NO:153 Protein sequence of LHA-EN-CPDY1-13-GS20-HT-   SEQ ID NO:154 Protein sequence of LHA-EN-CPDY-GS30-HT-   SEQ ID NO:155 Protein sequence of LHA-EN-CPDY13-GS30-HT-   SEQ ID NO:156 Protein sequence of LHA-EN-CPDY(D15A)-GS20-HT-   SEQ ID NO:157 Protein sequence of LHA-EN-CPDY(D15A)-GS30-HT-   SEQ ID NO:158 Protein sequence of LHB-EN-CPDY-HT-   SEQ ID NO:159 Protein sequence of LHA-EN-CPDYI8RP10RD15A-GS20-HT-   SEQ ID NO:160 Protein sequence of LHA-EN-CPDY(I8RP10R)1-13-GS20-HT-   SEQ ID NO:161 Protein sequence of LHA-EN-CPDY-HX27-HT-   SEQ ID NO:162 Protein sequence of LHA-EN-CPDNv9-HT-   SEQ ID NO:163 Protein sequence of LHA-Xa-CPNv-HT-   SEQ ID NO:164 Protein sequence of LHC-Xa-CPNv-HT-   SEQ ID NO:165 Protein sequence of LHD-EN-CPNv-HT-   SEQ ID NO:166 Protein sequence of LHA-Xa-CPN-HT-   SEQ ID NO:167 Protein sequence of LHB-EN-CPNv-HT-   SEQ ID NO:168 Protein sequence of LHA-CPOPv-HT-   SEQ ID NO:169 Protein sequence of LHA-Xa-GS-S6-ss

EXAMPLES Example 1 Confirmation of TM Agonist Activity by MeasuringRelease of Substance P from Neuronal Cell Cultures Materials

Substance P EIA is obtained from R&D Systems, UK.

Methods

Primary neuronal cultures of eDRG are established as describedpreviously (Duggan et al., 2002). Substance P release from the culturesis assessed by EIA, essentially as described previously (Duggan et al.,2002). The TM of interest is added to the neuronal cultures (establishedfor at least 2 weeks prior to treatment); control cultures are performedin parallel by addition of vehicle in place of TM. Stimulated (100 mMKCl) and basal release, together with total cell lysate content, ofsubstance P are obtained for both control and TM treated cultures.Substance P immunoreactivity is measured using to Substance P EnzymeImmunoassay Kits (Cayman Chemical Company, USA or R&D Systems, UK)according to manufacturers' instructions.

The amount of Substance P released by the neuronal cells in the presenceof the TM of interest is compared to the release obtained in thepresence and absence of 100 mM KCl. Stimulation of Substance P releaseby the TM of interest above the basal release, establishes that the TMof interest is an “agonist ligand” as defined in this specification. Ifdesired the stimulation of Substance P release by the TM of interest canbe compared to a standard Substance P release-curve produced using thenatural ORL-1 receptor ligand, nociceptin (Tocris).

Example 2 Expression and Purification of Catalytically Active LH_(N)/AMaterials

Synthetic DNA obtained from Sigma Genosys.

Restriction enzymes obtained from New England Biolabs.

Methods

The expression and purification of catalytically active LH_(N)/A wascarried out essentially as described in Sutton et al., (2005), Prot.Express. Purif., 40, pp 31-41.

Briefly, DNA encoding the light chain plus 423 amino acids from theN-terminal of the heavy chain of BoNT/A was synthesised by Sigma-Genosysto produce a synthetic LH_(N)/A gene with an E. coli codon bias. Thelinker region between the light chain and H_(N) domain was engineered tocontain a Factor Xa cleavage site by splice-overlap extension PCR. TwoPCR products were generated using primer pairs consisting of a long,mutagenic primer and a shorter, non-mutagenic primer:

(5′-tccaaaactaaatctctgATAGAAGGTAGAaacaaagcgctgaacgac; SEQ ID NO: 176)with (5′-CTTGATGTACTCTGTGAACGTGCTC; SEQ ID NO: 177); and(5′-gtcgttcagcgctttgttTCTACCTTCTATcagagatttagttttgga; SEQ ID N0: 178)with (5′-ATGGAGTTCGTTAACAAACAGTTC; SEQ ID NO: 179).

The products from these two reactions were used as templates for thesplice-overlap extension PCR. A further PCR reaction was set up to addBamHI and SalI sites at either end of the activatable recLH_(N)/A geneand these sites were used for insertion into an Invitrogen gateway entryvector. The entry vector was then used, along with a gatewayrecombination site adapted pMAL c2x, in a LR clonase reaction to formpMAL c2x recLH_(N)/A. The pMAL c2x recLH_(N)/A was modified toincorporate a 6′HIS tag at the N-terminus of the MBP. This was achievedby the insertion of annealed oligonucleotides encoding the HIS tag intothe NdeI site of pMAL.

The expression vector expressing LH_(N)/A was transformed into E. coliHMS174 or AD494 (DE3) (Novagen). Cultures were grown in Terrific brothcomplex medium supplemented with ZnCl₂ (1 μM), ampicillin (100 μg/ml),0.2% (w/v) glucose. Parameters for expression of all the constructs wereinitially determined in shake flask cultures before transferring into 8L fermentor systems. Starter cultures were grown for 16 hours at 37° C.,220 rpm and used to inoculate 1 L in which growth was continued at 37°C., 250 rpm. At an OD600 nm of 0.6 the temperature was reduced to 25° C.for 30 minutes before induction with 1 mM IPTG. Induction was continuedfor 4 hours before the cells were harvested and stored at −70° C.

Typically 16 g of cell paste was suspended in 160 ml PBS and lysed bysonication (MSE Soniprep 150). The resulting lysate was clarified bycentrifugation prior loading onto a 25 ml amylose column and eluted with10 mM maltose in PBS. The eluant contained approx. 50% pure fusionprotein and was treated with Factor Xa (1 unit Factor Xa/100 μg fusionprotein; 20 hours; 26° C.) to remove the HISMBP and cleave the LC-H_(N)junction to activate the protein. After incubation the sample wasfiltered (0.45 mm) and diluted two fold with water to give a 0.5×PBSbuffer composition. The cleaved, filtered and diluted recLH_(N)/A wasprocessed through a Q Sepharose FF column (10 ml) and eluted with a stepgradient of 80 mM NaCl containing HISMBP and 120 mM NaCl containingapprox. 75% pure recLH_(N)/A. The addition of His tag to MBP overcameprevious co-elution problems with LH_(N)/A and MBP. As a final polishingstep to ensure complete removal of the HISMBP, the 120 mM NaCl elutionfrom the Q Sepharose column was passed through a Nickel charged 5 mlHisTrap column (Amersham). The flow through from the HisTrap columncontained approx. 95% pure recLH_(N)/A (see the Figures in Sutton etal., (2005), Prot. Express. Purif., 40, pp 31-41 for an illustration ofthe purification scheme for LHN/A).

Example 3 Expression and Purification of Catalytically ActiveRecombinant LH_(N)/B

The methodology described below will purify catalytically activeLH_(N)/B protease from E. coli transformed with the appropriate plasmidencoding the LH_(N)/B polypeptide. It should be noted that varioussequences of suitable LH_(N)/B polypeptides have been described inPCT/GB97/02273, granted U.S. Pat. No. 6,461,617 and U.S. patentapplication Ser. No. 10/241,596, incorporated herein by reference.

Methods

The coding region for LH_(N)/B is inserted in-frame to the 3′ of thegene encoding maltose binding protein (MBP) in the expression vectorpMAL (New England Biolabs) to create pMAL-c2x-LH_(N)/B. In thisconstruct, the expressed MBP and LH_(N)/B polypeptides are separated bya Factor Xa cleavage site, and the LC and H_(N) domains are separated bya peptide that is susceptible to cleavage with enterokinase. Theexpression clone is termed pMAL-c2X-synLH_(N)/B.

pMAL-c2X-synLH_(N)/B is transformed into E. coli HMS174 and cultured inTerrific broth complex medium in 8 L fermentor systems. Pre-inductionbacterial growth is maintained at 37° C. to an OD600 nm of 5.0, at whichstage expression of recMBP-LH_(N)/B is induced by addition of IPTG to0.5 mM and a reduction in temperature to 30° C. After four hours at 30°C. the bacteria are harvested by centrifugation and the resulting pastestored at −70° C.

The cell paste is resuspended in 20 mM Hepes pH 7.2, 125 mM NaCl, 1 μMZnCl₂ and cell disruption achieved using an APV-Gaulin lab model 1000homogeniser or a MSE Soniprep 150 sonicator. The resulting suspension isclarified by centrifugation prior to purification.

Following cell disruption, the MBP-fusion protein is captured either onan amylose affinity resin in 20 mM Hepes pH 7.2, 125 mM NaCl, 1 μMZnCl₂, or on a Q-Sepharose FF anion-exchange resin in 50 mM Hepes pH7.2, 1 μM ZnCl₂ with no salt. A single peak is eluted from the amyloseresin in the same buffer plus 10 mM maltose and from the Q-Sepharose in150-200 mM salt. Cleavage of the MBP-LH_(N)/B junction is completed inan 18 hours incubation step at 22° C. with Factor Xa (NEB) at 1 U/50 μgfusion protein. A substrate (MBP-LH_(N)/B) concentration of at least 4mg/ml is desirable is for efficient cleavage to take place.

The cleaved protein is diluted with 20 mM Hepes to a buffer compositionof 20 mM Hepes, 25 mM NaCl, 1 μM ZnCl₂, pH 7.2 and processed through a QSepharose column to separate the MBP from LH_(N)/B. The LH_(N)/B iseluted from the Q-Sepharose column with 120-170 mM salt. The linkerbetween the light chain and H_(N) domain is then nicked by incubationwith enterokinase at 1 U/100 μg of LH_(N)/B at 22° C. for 16 hours.Finally, the enterokinase is separated from the nicked LH_(N)/B andother contaminating proteins on a Benzamidine Sepharose column, theenzyme preferentially binding to the resin over an incubation of 30minutes at 4° C. Purified LH_(N)/B is stored at −20° C. until required.See FIG. 1 for an illustration of the purification scheme forrecLH_(N)/B.

Example 4 Expression and Purification of Catalytically ActiveRecombinant LH_(N)/C

The coding region for LH_(N)/C is inserted in-frame to the 3′ of thegene encoding maltose binding protein (MBP) in the expression vectorpMAL (New England Biolabs) to create pMAL-c2x-LH_(N)/C. In thisconstruct the expressed MBP and LH_(N)/C polypeptides are separated by aFactor Xa cleavage site.

pMAL-c2x-LH_(N)/C is transformed into E. coli AD494 (DE3, IRL) andcultured in Terrific broth complex medium in 8 L fermentor systems.Pre-induction bacterial growth are maintained at 30° C. to an OD600 nmof 8.0, at which stage expression of recMBP-c2x-LH_(N)/C is induced byaddition of IPTG to 0.5 mM and a reduction in temperature of culture to25° C. After 4 hours at 25° C. the bacteria are harvested bycentrifugation and the resulting paste stored at −70° C.

The cell paste is resuspended in 50 mM Hepes pH 7.2, 1 μM ZnCl₂ at 1:6(w/v) and cell disruption is achieved using an APV-Gaulin lab model 1000homogeniser or a MSE Soniprep 150 sonicator. The resulting suspension isclarified by centrifugation prior to purification.

Following cell disruption and clarification, the MBP-fusion protein isseparated on a Q-Sepharose Fast Flow anion-exchange resin in 50 mM HepespH 7.2, 1 μM ZnCl₂ and eluted with the same buffer plus 100 mM NaCl. Adouble point cleavage is performed at the MBP-LH_(N)/C junction and theH_(N)-LC linker in a single incubation step with Factor Xa. The reactionis completed in a 16-hour incubation step at 22° C. with Factor Xa (NEB)at 1 U/100 ìg fusion protein. The cleaved protein is diluted with 20 mMHepes to a buffer composition of 20 mM Hepes, 25 mM NaCl, pH 7.2 andprocessed through a second Q-Sepharose column to separate the MBP fromLH_(N)/C. Activated (disulphide-bonded cleaved linker) LH_(N)/C iseluted from the Q-Sepharose column by a salt gradient (20 mM Hepes, 500mM NaCl, 1 μM ZnCl₂, pH 7.2) in 120-170 mM salt. See FIG. 2 for anillustration of the purification of LH_(N)/C.

Example 5 Production of a Chemical Conjugate of Nociceptin and LH_(N)/AMaterials

C-terminally extended nociceptin peptide obtained from Sigma Genosys.Conjugation chemicals obtained from Pierce.

Methods

In order to couple the nociceptin peptide via a C-terminal Cys, thepeptide was first synthesised (by standard procedures, commerciallyobtainable) to include a Cys as the final C-terminal amino acid.

This peptide was then used as the second component in a sulphydryl basedcoupling reaction as described below (see also previous publications WO99/17806 and WO 96/33273 and Duggan et al., (2002), J. Biol. Chem. 277,24846-34852 and Chaddock et al., (2000), Infect Immun., 68, 2587-2593).

Sulphydryl Based Coupling Reaction

Briefly, approximately two reactive leaving groups were introduced intoLH_(N)/A (5 mg/ml in phosphate-buffered saline) by reaction withN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP).

Derivatised material was isolated from excess SPDP by size exclusionchromatography. Reconstituted cysteine-tagged nociceptin ligand wasmixed with the derivatised LH_(N)/A in a 4:1 molar ratio, and incubatedat room temperature for 1 hour with gentle agitation is in order tocreate a chemical conjugate through a reducible covalent disulphidebond.

Initial fractionation of the conjugate mixture to remove unconjugatedpeptide was performed by size exclusion chromatography (Superose-12, orSuperdex G-200 depending on scale of conjugation).

Example 6 Production of a Chemical Conjugate of Nociceptin and LH_(N)/BMaterials

C-terminally extended nociceptin peptide obtained from Sigma Genosys.

Conjugation chemicals obtained from Pierce.

Methods

Lyophilised nociceptin was dissolved by the addition of water anddialysed into MES buffer (0.1 M MES, 0.1 M NaCl, pH 5.0). To thissolution (at a concentration of about 0.3 mg/ml) was added PDPH (100mg/ml in DMF) to a final concentration of 1 mg/ml. After mixing, solidEDAC was added to produce a final concentration of about 0.2 mg/ml. Thereaction was allowed to proceed for at least 30 minutes at roomtemperature. Excess PDPH was then removed by desalting over a PD-10column (Pharmacia) previously equilibrated with MES buffer.

An amount of LH_(N)/B equivalent to half the weight of nociceptin useddissolved in triethanolamine buffer (0.02 M triethanolamine/HCl, 0.1 Msodium chloride, pH 7.8) at a concentration of about 1 mg/ml, wasreacted with Traut's reagent (100 mM stock solution in 1 Mtriethanolamine/HCl, pH 8.0) at a final concentration of 2 mM. After 1hour, the LH_(N)/B was desalted into PBSE (phosphate buffered salinewith 1 mM EDTA) using a PD-10 column (Pharmacia). The protein peak fromthe column eluate was concentrated using a Microcon 50 (Amicon) to aconcentration of about 2 mg/ml.

The derivatised nociceptin was subjected to a final concentration stepresulting in a reduction in volume to less than 10% of the startingvolume and then mixed with the derivatised LH_(N)/B overnight at roomtemperature. The products of the reaction were analysed bypolyacrylamide gel electrophoresis in the presence of sodiumdodecyl-sulphate (SDS-PAGE).

The conjugate resulting from the above reaction was partially purifiedby size exclusion chromatography over Bio-Gel P-100 (BioRad). Theelution profile was followed by measuring the optical density at 280 nmand SDS-PAGE analysis of the fractions. This allowed the separation ofconjugate from free nociceptin and by-products of the reaction.

Example 7 Production of a Chemical Conjugate of Nociceptin 1-11 andLH_(N)/B Materials

C-terminally extended nociceptin 1-11 peptide obtained from SigmaGenosys.

Conjugation chemicals obtained from Pierce.

Methods

In order to couple the nociceptin 1-11 peptide via a C-terminal Cys, thepeptide was first synthesised (by standard procedures, commerciallyobtainable) to include a Cys as the final C-terminal amino acid.

This peptide was then used as the second component in a sulphydryl basedcoupling reaction as described in Example 5.

Example 8 Production of a Chemical Conjugate of Nociceptin N[[Y14]1-17]and LH_(N)/C Materials

C-terminally extended nociceptin N[[Y14]1-17] peptide obtained fromSigma Genosys. Conjugation chemicals obtained from Pierce.

Methods

In order to couple the peptide via a C-terminal Cys, the peptide wasfirst synthesised (by to standard procedures, commercially obtainable)to include a Cys as the final C-terminal amino acid.

This peptide was then used as the second component in a sulphydryl basedcoupling reaction as described in Example 5.

Example 9 Recombinant Production of a Single Polypeptide Fusion ofNociceptin-LH_(N)/A (SEQ ID NO:15 and SEQ ID NO:16)

The DNA sequence for the nociceptin-LH_(N)/A was designed by backtranslation of the LC/A, H_(N)/A, and nociceptin amino acid sequences.The complete ORF containing the nociceptin-LC/A-activation loop-H_(N)/Asequence was assembled within standard DNA sequence manipulationsoftware (EditSeq). The activation loop between the LC/A cysteine andthe H_(N)/A cysteine (CVRGIITSKTKSLDKGYNKALNDLC; SEQ ID NO:180) wasmodified to incorporate a Factor Xa protease recognition site.

Restriction sites appropriate to facilitate cloning into the requiredexpression vector (for example BamHI/SalI) were incorporated at the 5′and 3′ ends respectively of the sequence maintaining the correct readingframe. The DNA sequence was screened (using software such as MapDraw,DNASTAR Inc.) for restriction enzyme cleavage sequences incorporatedduring the back translation. Any cleavage sequences that were found tobe common to those required by the cloning system were removed manuallyfrom the proposed coding sequence ensuring common E. coli codon usagewas maintained. E. coli codon usage was assessed by reference tosoftware programs such as Graphical Codon Usage Analyser (Geneart), andthe % GC content and codon usage ratio assessed by reference topublished codon usage tables (for example GenBank Release 143, 13 Sep.2004).

This optimised DNA sequence containing the nociceptin-LC/A-activationloop-H_(N)/A open reading frame (ORF) was then commercially synthesizedand provided in the pCR 4 vector.

The DNA encoding the nociceptin-LH_(N)/A fusion was isolated from pCR 4and transferred into pMAL vector backbone to facilitate proteinexpression. The resultant pMAL NO-LHN/A vector was transformed intocompetent E. coli BL21 and correct transformants selected. A singlecolony of pMAL NO-LH_(N)/A was grown in Terrific broth complex mediumsupplemented with ZnCl₂ (1 mM), ampicillin (100 μg/ml), 0.2% (w/v)glucose. Expression of the insert was induced by the addition of IPTG(0.1 mM) and the culture maintained at 16° C. for 16 hours. After thisperiod of expression the bacteria were isolated by centrifugation andthe cell pellet stored at −20° C. until use.

10 g of E. coli BL21 cell paste was defrosted in a falcon tubecontaining 25 ml 50 mM HEPES, pH 7.2, 200 mM NaCl. The thawed cell pastewas made up to 80 ml with 50 mM HEPES, pH 7.2, 200 mM NaCl and sonicatedon ice 30 seconds on, 30 seconds off for 10 cycles at a power of 22microns ensuring the sample remained cool. The lysed cells werecentrifuged at 18 000 rpm, 4° C. for 30 minutes. The supernatant wasloaded onto a 0.1 M NiSO₄ charged chelating column (20-30 ml column issufficient) and equilibrated with 50 mM HEPES, pH 7.2, 200 mM NaCl.

Using a step gradient of 10 and 40 mM imidazol, the non-specific boundprotein was washed away and the fusion protein eluted with 100 mMimidazol. The eluted fusion protein was dialysed against 5 L of 50 mMHEPES, pH 7.2, 200 mM NaCl at 4° C. overnight and the OD of the dialysedfusion protein measured. 1 unit of Factor Xa was added per 100 μg fusionprotein and incubated at 25° C. static overnight. The cleavage mixturewas loaded onto a 0.1 M NiSO₄ charged Chelating column (20-30 ml columnis sufficient) and equilibrated with 50 mM HEPES, pH 7.2, 200 mM NaCl.

Using a step gradient of 10 and 40 mM imidazol, the non-specific boundprotein was washed away and the fusion protein eluted with 100 mMimidazol. The eluted fusion protein was dialysed against 5 L of 50 mMHEPES, pH 7.2, 200 mM NaCl at 4° C. overnight and the fusionconcentrated to about 2 mg/ml, aliquoted and stored at −20° C.

FIG. 3 shows the SDS-PAGE analysis of expression and purification ofN[1-17]-LH_(N)/A

Example 10 Recombinant Production of a Single Polypeptide Fusion of(Nociceptin 1-11)-LH_(N)/B

The DNA sequence for the (nociceptin 1-11)-LH_(N)/B was designed by backtranslation of the LC/B, H_(N)/B, and nociceptin 1-11 amino acidsequences. The complete ORF containing the(nociceptin1-11)-LC/B-activation loop-H_(N)/B sequence was assembledwithin standard DNA sequence manipulation software (EditSeq). Theactivation loop is between the LC/B cysteine and the H_(N)/B cysteinewas modified to incorporate a Factor Xa protease recognition site.

The recombinant fusion protein was then produced essentially asdescribed in Example 9.

Example 11 Recombinant Production of a Single Polypeptide Fusion of(Nociceptin N[[Y14]1-17])-LH_(N)/C (SEQ ID NO:25 and SEQ ID NO:26)

The DNA sequence for the nociceptin N[[Y14]1-17] was designed by backtranslation of the LC/C, H_(N)/C, and nociceptin N[[Y14]1-17] amino acidsequences. The complete ORF containing the (nociceptinN[[Y14]1-17])-LC/C-activation loop-H_(N)/C sequence was assembled withinstandard DNA sequence manipulation software (EditSeq). The activationloop between the LC/C cysteine and the H_(N)/C cysteine was modified toincorporate a Factor Xa protease recognition site.

The recombinant fusion protein was then produced essentially asdescribed in Example 9.

Example 12 Recombinant Production of a Single Polypeptide Fusion ofLH_(N)/C-(Nociceptin 1-11) (SEQ ID NO:23 and SEQ ID NO:24)

The DNA sequence for the LH_(N)/C-(nociceptin 1-11) was designed by backtranslation of the LC/C, H_(N)/C and nociceptin 1-11 amino acidsequences. The complete ORF (SEQ ID NO:23) containing theLC/C-activation loop-H_(N)/C-flexible spacer-(nociceptin 1-11) wasassembled within standard DNA sequence manipulation software (EditSeq).

The recombinant fusion protein (SEQ ID NO:24) was then producedessentially as described in Example 9.

Example 13 Production of a Conjugate for Delivery of DNA encoding LC/Cinto a Cell

The construction of a nociceptin-H_(N)-[LC/C] conjugate is describedbelow, where [LC/C] represents the polylysine condensed DNA encoding thelight chain of botulinum neurotoxin type C.

Materials

SPDP is from Pierce Chemical Co.

Additional reagents are obtained from Sigma Ltd.

Methods

Using a plasmid containing the gene encoding LC/C under the control of aCMV (immediate early) promoter, condensation of DNA was achieved usingSPDP-derivatised polylysine to a ratio of 2 DNA to 1 polylysine.Conjugates were then prepared by mixing condensed DNA (0.4 mg/ml) withH_(N)-nociceptin (100 μg/ml) for 16 h at 25° C. The SPDP-derivatisedpolylysine and the free —SH group present on the H_(N) domain combine tofacilitate covalent attachment of the DNA and protein.

Example 14 Production of a Conjugate for Delivery of DNA Encoding LC/Binto a Cell

The construction of a (nociceptin 1-11)-H_(N)-[LC/B] conjugate isdescribed below, where [LC/B] represents the polylysine condensed DNAencoding the light chain of botulinum neurotoxin type B.

Materials

SPDP is from Pierce Chemical Co.

Additional reagents are obtained from Sigma Ltd.

Methods

Using a plasmid containing the gene encoding LC/B under the control of aCMV (immediate early) promoter, condensation of DNA was achieved usingSPDP-derivatised polylysine to a ratio of 2 DNA to 1 polylysine.Conjugates were then prepared by mixing condensed DNA (0.4 mg/ml) withH_(N)-(nociceptin 1-11) (100 μg/ml) for 16 h at 25° C. TheSPDP-derivatised polylysine and the free —SH group present on the H_(N)domain combine to facilitate covalent attachment of the DNA and protein.

Example 15 Assessment of the Activity of Nociceptin-LH_(N)/A inSubstance P Releasing Neuronal Cells

Using methodology described in Duggan et al., (2002, J. Biol. Chem.,277, 34846-34852), the activity of nociceptin-LH_(N)/A in substance Preleasing neuronal cells was assessed.

Nociceptin-LH_(N)/A fusion protein was applied to 2-week old dorsal rootganglia neuronal cultures, and incubated at 37° C. for 16 hours.Following the incubation, the media was removed and the ability of thecells to undergo stimulated release of substance P(SP) was assessed.

The release of SP from the neuronal cells incubated with thenociceptin-LH_(N)/A fusion protein was assayed in comparison to (i)LH_(N)/A-only treated cells and (ii) cells treated with media alone.This allowed the % inhibition of substance P from the eDRG to becalculated. The ability of the nociceptin-LH_(N)/A fusion protein toinhibit SP release (relative to cells treated with media alone) wasreported in Table 1. The data represent the mean of 3 determinations:

TABLE 1 Nociceptin-LH_(N)/A fusion protein LH_(N)/A-only Test Material(μM) % Inhibition % Inhibition 1.0 47.3 25.6 0.1 13.8 −11.5

Example 16 Confirmation of ORL₁ Receptor Activation by MeasuringForskolin-Stimulated cAMP Production

Confirmation that a given TM is acting via the ORL₁ receptor is providedby the following test, in which the TMs ability to inhibitforskolin-stimulated cAMP production is assessed.

Materials

[³H]adenine and [¹⁴C]cAMP are obtained from GE Healthcare

Methods

The test is conducted essentially as described previously by Meunier etal. [Isolation and structure of the endogenous agonist of opioidreceptor-like ORL₁ receptor. Nature 377: 532-535, 1995] in intacttransfected-CHO cells plated on 24-well plastic plates.

To the cells is added [3H]adenine (1.0 μCi) in 0.4 ml of culture medium.The cells remain at 37° C. for 2 h to allow the adenine to incorporateinto the intracellular ATP pool. After 2 h, the cells are washed oncewith incubation buffer containing: 130 mM NaCl, 4.8 mM KCl, 1.2 mMKH₂PO₄, 1.3 mM CaCl₂, 1.2 mM MgSO₄, 10 mM glucose, 1 mg/ml bovine serumalbumin and 25 mM HEPES, pH 7.4, and replaced with buffer containingforskolin (10 μM) and isobutylmethylxanthine (50 μM) with or without theTM of interest. After 10 min., the medium is aspirated and replaced with0.5 ml, 0.2 M HCl. Approximately 1000 cpm of [¹⁴C]cAMP is added to eachwell and used as an internal standard. The contents of the wells arethen transferred to columns of 0.65 g dry alumina powder. The columnsare eluted with 4 ml of 5 mM HCl, 0.5 ml of 0.1 M ammonium acetate, thentwo additional millilitres of ammonium acetate. The final eluate iscollected into scintillation vials and counted for ¹⁴C and tritium.Amounts collected are corrected for recovery of [¹⁴C]cAMP. TMs that areagonists at the ORL₁ receptor cause a reduction in the level of cAMPproduced in response to forskolin.

Example 17 Confirmation of ORL₁ Receptor Activation Using a GTPγSBinding Functional Assay

Confirmation that a given TM is acting via the ORL₁ receptor is alsoprovided by the following test, a GTPγS binding functional assay.

Materials

[³⁵S]GTPγS is obtained from GE Healthcare

Wheatgerm agglutinin-coated (SPA) beads are obtained from GE Healthcare

Methods

This assay is carried out essentially as described by Traynor andNahorski [Modulation by β-opioid agonists ofguanosine-5-O-(3-[³⁵S]thio)triphosphate binding to membranes from humanneuroblastoma SH-SY5Y cells. Mol. Pharmacol. 47: 848-854, 1995].

Cells are scraped from tissue culture dishes into 20 mM HEPES, 1 mMethylenediaminetetraacetic acid, then centrifuged at 500×g for 10 min.Cells are resuspended in this buffer and homogenized with a PolytronHomogenizer.

The homogenate is centrifuged at 27,000×g for 15 min., and the pelletresuspended in buffer A, containing: 20 mM HEPES, 10 mM MgCl₂, 100 mMNaCl, pH 7.4. The suspension is recentrifuged at 20,000×g and suspendedonce more in buffer A. For the binding assay, membranes (8-15 μgprotein) are incubated with [³⁵S]GTP S (50 pM), GDP (10 μM), with andwithout the TM of interest, in a total volume of 1.0 ml, for 60 min. at25° C. Samples are filtered over glass fibre filters and counted asdescribed for the binding assays.

Example 18 Preparation of a LC/A and H_(N)/A Backbone Clones

The following procedure creates the LC and H_(N) fragments for use asthe component backbone for multidomain fusion expression. This exampleis based on preparation of a serotype A based clone (SEQ ID NO:27 andSEQ ID NO:28), though the procedures and methods are equally applicableto the other serotypes [illustrated by the sequence listing for serotypeB (SEQ ID NO:29 and SEQ ID NO:30) and serotype C (SEQ ID NO:31 and SEQID NO:32)].

Preparation of Cloning and Expression Vectors

pCR 4 (Invitrogen) is the chosen standard cloning vector, selected dueto the lack of restriction sequences within the vector and adjacentsequencing primer sites for easy construct confirmation. The expressionvector is based on the pMAL (NEB) expression vector, which has thedesired restriction sequences within the multiple cloning site in thecorrect orientation for construct insertion (BamHI-SalI-PstI-HindIII). Afragment of the expression vector has been removed to create anon-mobilisable plasmid and a variety of different fusion tags have beeninserted to increase purification options.

Preparation of Protease (e.g. LC/a) Insert

The LC/A (SEQ ID NO:27) is created by one of two ways:

The DNA sequence is designed by back translation of the LC/A amino acidsequence [obtained from freely available database sources such asGenBank (accession number P10845) or Swissprot (accession locusBXA1_CLOBO) using one of a variety of reverse translation software tools(for example EditSeq best E. coli reverse translation (DNASTAR Inc.), orBacktranslation tool v2.0 (Entelechon)]. BamHI/SalI recognitionsequences are incorporated at the 5′ and 3′ ends respectively of thesequence, maintaining the correct reading frame. The DNA sequence isscreened (using software such as MapDraw, DNASTAR Inc.) for restrictionenzyme cleavage sequences incorporated during the back translation. Anycleavage sequences that are found to be common to those required by thecloning system are removed manually from the proposed coding sequenceensuring common E. coli codon usage is maintained. E. coli codon usageis assessed by reference to software programs such as Graphical CodonUsage Analyser (Geneart), and the % GC content and codon usage ratioassessed by reference to published codon usage tables (for exampleGenBank Release 143, 13 Sep. 2004). This optimised DNA sequencecontaining the LC/A open reading frame (ORF) is then commerciallysynthesized (for example by Entelechon, Geneart or Sigma-Genosys) and isprovided in the pCR 4 vector.

The alternative method is to use PCR amplification from an existing DNAsequence with BamHI and SalI restriction enzyme sequences incorporatedinto the 5′ and 3′ PCR primers respectively. Complementaryoligonucleotide primers are chemically synthesised by a supplier (forexample MWG or Sigma-Genosys), so that each pair has the ability tohybridize to the opposite strands (3′ ends pointing “towards” eachother) flanking the stretch of Clostridium target DNA, oneoligonucleotide for each of the two DNA strands. To generate a PCRproduct the pair of short oligonucleotide primers specific for theClostridium DNA sequence are mixed with the Clostridium DNA template andother reaction components and placed in a machine (the ‘PCR machine’)that can change the incubation temperature of the reaction tubeautomatically, cycling between approximately 94° C. (for denaturation),55° C. (for oligonucleotide annealing), and 72° C. (for synthesis).Other reagents required for amplification of a PCR product include a DNApolymerase (such as Taq or Pfu polymerase), each of the four nucleotidedNTP building blocks of DNA in equimolar amounts (50-200 μM) and abuffer appropriate for the enzyme optimised for Mg²⁺ concentration(0.5-5 mM).

The amplification product is cloned into pCR 4 using either, TOPO TAcloning for Taq PCR products or Zero Blunt TOPO cloning for Pfu PCRproducts (both kits commercially available from Invitrogen). Theresultant clone is checked by sequencing. Any additional restrictionsequences which are not compatible with the cloning system are thenremoved using site directed mutagenesis [for example, using Quickchange(Stratagene Inc.)].

Preparation of Translocation (e.g. H_(N)) InsertThe H_(N)/A (SEQ ID NO:28) is created by one of two ways:

The DNA sequence is designed by back translation of the H_(N)/A aminoacid sequence [obtained from freely available database sources such asGenBank (accession number P10845) or Swissprot (accession locusBXA1_CLOBO)] using one of a variety of reverse translation softwaretools [for example EditSeq best E. coli reverse translation (DNASTARInc.), or Backtranslation tool v2.0 (Entelechon)]. A PstI restrictionsequence added to the N-terminus and XbaI-stop codon-HindIII to theC-terminus ensuring the correct reading frame is maintained. The DNAsequence is screened (using software such as MapDraw, DNASTAR Inc.) forrestriction enzyme cleavage sequences incorporated during the backtranslation. Any sequences that are found to be common to those requiredby the cloning system are removed manually from the proposed codingsequence ensuring common E. coli codon usage is maintained. E. colicodon usage is assessed by reference to software programs such asGraphical Codon Usage Analyser (Geneart), and the % GC content and codonusage ratio assessed by reference to published codon usage tables (forexample GenBank Release 143, 13 Sep. 2004). This optimised DNA sequenceis then commercially synthesized (for example by Entelechon, Geneart orSigma-Genosys) and is provided in the pCR 4 vector.

The alternative method is to use PCR amplification from an existing DNAsequence with PstI and XbaI-stop codon-HindIII restriction enzymesequences incorporated into the 5′ and 3′ PCR primers respectively. ThePCR amplification is performed as described above. The PCR product isinserted into pCR 4 vector and checked by sequencing. Any additionalrestriction sequences which are not compatible with the cloning systemare then removed using site directed mutagenesis [for example usingQuickchange (Stratagene Inc.)].

Example 19 Preparation of a LC/A-Nociceptin-H_(N)/A Fusion Protein(Nociceptin is N-Terminal of the H_(N)-Chain) Preparation ofLinker-Nociceptin-Spacer Insert

The LC-H_(N) linker can be designed from first principle, using theexisting sequence information for the linker as the template. Forexample, the serotype A linker (in this case defined as the inter-domainpolypeptide region that exists between the cysteines of the disulphidebridge between LC and H_(N)) is 23 amino acids long and has the sequenceVRGIITSKTKSLDKGYNKALNDL (amino acids 2-24 of SEQ ID NO:180). Within thissequence, it is understood that proteolytic activation in nature leadsto an H_(N) domain that has an N-terminus of the sequence ALNDL. Thissequence information is freely available from available database sourcessuch as GenBank (accession number P10845) or Swissprot (accession locusBXA1_CLOBO). Into this linker a Factor Xa site, nociceptin and spacerare incorporated; and using one of a variety of reverse translationsoftware tools [for example EditSeq best E. coli reverse translation(DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)], the DNAsequence encoding the linker-ligand-spacer region is determined.Restriction sites are then incorporated into the DNA sequence and can bearranged as BamHI-SalI-linker-proteasesite-nociceptin-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII (SEQ IDNO:33). It is important to ensure the correct reading frame ismaintained for the spacer, nociceptin and restriction sequences and thatthe XbaI sequence is not preceded by the bases, TC, which would resulton DAM methylation. The DNA sequence is screened for restrictionsequence incorporation, and any additional sequences are removedmanually from the remaining sequence ensuring common E. coli codon usageis maintained. E. coli codon usage is assessed by reference to softwareprograms such as Graphical Codon Usage Analyser (Geneart), and the % GCcontent and codon usage ratio assessed by reference to published codonusage tables (for example, GenBank Release 143, 13 Sep. 2004). Thisoptimised DNA sequence is then commercially synthesized (for example byEntelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4vector.

Preparation of the LC/A-Nociceptin-H_(N)/A Fusion

In order to create the LC-linker-nociceptin-spacer-H_(N) construct (SEQID NO:39), the pCR 4 vector encoding the linker (SEQ ID NO:33) iscleaved with BamHI+SalI restriction enzymes. This cleaved vector thenserves as the recipient vector for insertion and ligation of the LC/ADNA (SEQ ID NO:27) cleaved with BamHI+SalI. The resulting plasmid DNA isthen cleaved with PstI+XbaI restriction enzymes and serves as therecipient vector for the insertion and ligation of the H_(N)/A DNA (SEQID NO:28) cleaved with PstI+XbaI. The final construct contains theLC-linker-nociceptin-spacer-H_(N) ORF (SEQ ID NO:39) for transfer intoexpression vectors for expression to result in a fusion protein of thesequence illustrated in SEQ ID NO:40.

Example 20 Preparation of a Nociceptin-LC/A-H_(N)/A Fusion Protein(Nociceptin is N-Terminal of the LC-Chain)

The LC/A-H_(N)/A backbone is constructed as described in Example 19using the synthesised A serotype linker with the addition of a Factor Xasite for activation, arranged as BamHI-SalI-linker-proteasesite-linker-PstI-XbaI-stop codon-HindIII (SEQ ID NO:34). TheLC/A-H_(N)/A backbone and the synthesised N-terminal presentationnociceptin insert (SEQ ID NO:35) are cleaved with BamHI+HindIIIrestriction enzymes, gel purified and ligated together to create anociceptin-spacer-LC-linker-H_(N). The ORF (SEQ ID NO:41) is then cutout using restriction enzymes AvaI+XbaI for transfer into expressionvectors for expression to result in a fusion protein of the sequenceillustrated in SEQ ID NO:42.

Example 21 Preparation of a LC/C-Nociceptin-H_(N)/C Fusion Protein

Following the methods used in Examples 1 and 2, the LC/C (SEQ ID NO:31)and H_(N)/C (SEQ ID NO:32) are created and inserted into the C serotypelinker arranged as BamHI-Sail-linker-proteasesite-nociceptin-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII (SEQ IDNO:36). The final construct contains theLC-linker-nociceptin-spacer-H_(N) ORF (SEQ ID NO:43) for expression as aprotein of the sequence illustrated in SEQ ID NO:44.

Example 22 Preparation of a LC/C-Nociceptin-H_(N)/C Fusion Protein witha Serotype A Activation Sequence

Following the methods used in Examples 1 and 2, the LC/C (SEQ ID NO:31)and H_(N)/C (SEQ ID NO:32) are created and inserted into the A serotypelinker arranged as BamHI-Sail-linker-proteasesite-nociceptin-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII (SEQ IDNO:33). The final construct contains theLC-linker-nociceptin-spacer-H_(N) ORF (SEQ ID NO:45) for expression as aprotein of the sequence illustrated in SEQ ID NO:46.

Example 23 Preparation of a LC/A-Met Enkephalin-H_(N)/A Fusion Protein

Due to the small, five-amino acid, size of the met-enkephalin ligand theLC/A-met enkephalin-H_(N)/A fusion is created by site directedmutagenesis [for example using Quickchange (Stratagene Inc.)] using theLC/A-nociceptin-H_(N)/A fusion (SEQ ID NO:39) as a template.Oligonucleotides are designed encoding the YGGFM met-enkephalin peptide(SEQ ID NO:181), ensuring standard E. coli codon usage is maintained andno additional restriction sites are incorporated, flanked by sequencescomplimentary to the linker region of the LC/A-nociceptin-H_(N)/A fusion(SEQ ID NO:39) either side on the nociceptin section. The SDM product ischecked by sequencing and the final construct containing theLC-linker-met enkephalin-spacer-H_(N) ORF (SEQ ID NO:47) for expressionas a protein of the sequence illustrated in SEQ ID NO:48.

Example 24 Preparation of a LC/A-β Endorphin-H_(N)/A Fusion Protein

Following the methods used in Examples 1 and 2, the LC/A (SEQ ID NO:27)and H_(N)/A (SEQ ID NO:28) are created and inserted into the A serotype13 endorphin linker arranged as BamHI-Sail-linker-protease site-βendorphin-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII (SEQ ID NO:37).The final construct contains the LC-linker-β endorphin-spacer-H_(N) ORF(SEQ ID NO:49) for expression as a protein of the sequence illustratedin SEQ ID NO:50.

Example 25 Preparation of a LC/A-Nociceptin Variant-H_(N)/A FusionProtein

Following the methods used in Examples 1 and 2, the LC/A (SEQ ID NO:27)and H_(N)/A (SEQ ID NO:28) are created and inserted into the A serotypenociceptin variant linker arranged as BamHI-Sail-linker-proteasesite-nociceptin variant-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII(SEQ ID NO:38). The final construct contains the LC-linker-nociceptinvariant-spacer-H_(N) ORF (SEQ ID NO:51) for expression as a protein ofthe sequence illustrated in SEQ ID NO:52.

Example 26 Purification Method for LC/A-Nociceptin-H_(N)/A FusionProtein

Defrost falcon tube containing 25 ml 50 mM HEPES pH 7.2, 200 mM NaCl andapproximately 10 g of E. coli BL21 cell paste. Make the thawed cellpaste up to 80 ml with 50 mM HEPES pH 7.2, 200 mM NaCl and sonicate onice 30 seconds on, 30 seconds off for 10 cycles at a power of 22 micronsensuring the sample remains cool. Spin the lysed cells at 18 000 rpm, 4°C. for 30 minutes. Load the supernatant onto a 0.1 M NiSO₄ chargedChelating column (20-30 ml column is sufficient) equilibrated with 50 mMHEPES pH 7.2, 200 mM NaCl. Using a step gradient of 10 and 40 mMimidazol, wash away the non-specific bound protein and elute the fusionprotein with 100 mM imidazol. Dialyse the eluted fusion protein against5 L of 50 mM HEPES pH 7.2, 200 mM NaCl at 4° C. overnight and measurethe OD of the dialysed fusion protein. Add 1 unit of factor Xa per 100μg fusion protein and Incubate at 25° C. static overnight. Load onto a0.1 M NiSO₄ charged Chelating column (20-30 ml column is sufficient)equilibrated with 50 mM HEPES pH 7.2, 200 mM NaCl. Wash column tobaseline with 50 mM HEPES pH 7.2, 200 mM NaCl. Using a step gradient of10 and 40 mM imidazol, wash away the non-specific bound protein andelute the fusion protein with 100 mM imidazol. Dialyse the eluted fusionprotein against 5 L of 50 mM HEPES pH 7.2, 200 mM NaCl at 4° C.overnight and concentrate the fusion to about 2 mg/ml, aliquot sampleand freeze at −20° C. Test purified protein using OD, BCA, purityanalysis and SNAP-25 assessments.

Example 27 Preparation of a LC/A-Nociceptin-H_(N)/A Fusion Protein(Nociceptin is N-Terminal of the H_(N)-Chain)

The linker-nociceptin-spacer insert is prepared as described in Example19.

Preparation of the LC/A-Nociceptin-H_(N)/A fusion

In order to create the LC-linker-nociceptin-spacer-H_(N) construct (SEQID NO:39), the pCR 4 vector encoding the linker (SEQ ID NO:33) iscleaved with BamHI+SalI restriction enzymes. This cleaved vector thenserves as the recipient for insertion and ligation of the LC/A DNA (SEQID NO:27) also cleaved with BamHI+SalI. The resulting plasmid DNA isthen cleaved with BamHI+HindIII restriction enzymes and the LC/A-linkerfragment inserted into a similarly cleaved vector containing a uniquemultiple cloning site for BamHI, SalI, PstI, and HindIII such as thepMAL vector (NEB). The H_(N)/A DNA (SEQ ID NO:28) is then cleaved withPstI+HindIII restriction enzymes and inserted into the similarly cleavedpMAL-LC/A-linker construct. The final construct contains theLC-linker-nociceptin-spacer-H_(N) ORF (SEQ ID NO:39) for expression as aprotein of the sequence illustrated in SEQ ID NO:40.

Example 28 Preparation of a Nociceptin-LC/A-H_(N)/A Fusion Protein(Nociceptin is N-Terminal of the LC-Chain)

In order to create the nociceptin-spacer-LC/A-H_(N)/A construct, an Aserotype linker with the addition of a Factor Xa site for activation,arranged as BamHI-Sail-linker-protease site-linker-PstI-XbaI-stopcodon-HindIII (SEQ ID NO:34) is synthesised as described in Example 27.The pCR 4 vector encoding the linker is cleaved with BamHI+SalIrestriction enzymes. This cleaved vector then serves as the recipientfor insertion and ligation of the LC/A DNA (SEQ ID NO:27) also cleavedwith BamHI+SalI. The resulting plasmid DNA is then cleaved withBamHI+HindIII restriction enzymes and the LC/A-linker fragment insertedinto a similarly cleaved vector containing the synthesised N-terminalpresentation nociceptin insert (SEQ ID NO:35). This construct is thencleaved with AvaI+HindIII and inserted into an expression vector such asthe pMAL plasmid (NEB). The H_(N)/A DNA (SEQ ID NO:28) is then cleavedwith PstI+HindIII restriction enzymes and inserted into the similarlycleaved pMAL-nociceptin-LC/A-linker construct. The final constructcontains the nociceptin-spacer-LC/A-H_(N)/A ORF (SEQ ID NO:63) forexpression as a protein of the sequence illustrated in SEQ ID NO:64.

Example 29 Preparation and Purification of an LC/A-Nociceptin-H_(N)/AFusion Protein Family with Variable Spacer Length

Using the same strategy as employed in Example 19, a range of DNAlinkers were prepared that encoded nociceptin and variable spacercontent. Using one of a variety of reverse translation software tools[for example EditSeq best E. coli reverse translation (DNASTAR Inc.), orBacktranslation tool v2.0 (Entelechon)], the DNA sequence encoding thelinker-ligand-spacer region is determined. Restriction sites are thenincorporated into the DNA sequence and can be arranged asBamHI-SalI-linker-proteasesite-nociceptin-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII (SEQ IDNO:65 to SEQ ID NO:69). It is important to ensure the correct readingframe is maintained for the spacer, nociceptin and restriction sequencesand that the XbaI sequence is not preceded by the bases, TC which wouldresult on DAM methylation. The DNA sequence is screened for restrictionsequence incorporation and any additional sequences are removed manuallyfrom the remaining sequence ensuring common E. coli codon usage ismaintained. E. coli codon usage is assessed by reference to softwareprograms such as Graphical Codon Usage Analyser (Geneart), and the % GCcontent and codon usage ratio assessed by reference to published codonusage tables (for example GenBank Release 143, 13 Sep. 2004). Thisoptimised DNA sequence is then commercially synthesized (for example byEntelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4vector.

The spacers that were created included:

TABLE 2 SEQ ID NO:  of the Code Protein sequence of the linker linker DNA GS10 ALAGGGGSALVLQ 182 GS15 ALAGGGGSGGGGSALVLQ 183 GS25ALAGGGGSGGGGSGGGGSGGGGSALVLQ 184 GS30 ALAGGGGSGGGGSGGGGSGGGGSGGGGSALVLQ185 HX27 ALAAEAAAKEAAAKEAAAKAGGGGSALVLQ 186

By way of example, in order to create the LC/A-CPN(GS15)-H_(N)/A fusionconstruct (SEQ ID NO:70), the pCR 4 vector encoding the linker (SEQ IDNO:66) is cleaved with BamHI+SalI restriction enzymes. This cleavedvector then serves as the recipient vector for insertion and ligation ofthe LC/A DNA (SEQ ID NO:27) also cleaved with BamHI+SalI. The resultingplasmid DNA is then cleaved with BamHI+HindIII restriction enzymes andthe LC/A-linker fragment inserted into a similarly cleaved vectorcontaining a unique multiple cloning site for BamHI, SalI, PstI, andHindIII such as the pMAL vector (NEB). The H_(N)/A DNA (SEQ ID NO:28) isthen cleaved with PstI+HindIII restriction enzymes and inserted into thesimilarly cleaved pMAL-LC/A-linker construct. The final constructcontains the LC/A-CPN(GS15)-H_(N)/A ORF (SEQ ID NO:70) for expression asa protein of the sequence illustrated in SEQ ID NO:71.

As a further example, to create the LC/A-CPN(GS25)-H_(N)/A fusionconstruct (SEQ ID NO:72), the pCR 4 vector encoding the linker (SEQ IDNO:67) is cleaved with BamHI+SalI restriction enzymes. This cleavedvector then serves as the recipient vector for insertion and ligation ofthe LC/A DNA (SEQ ID NO:27) cleaved with BamHI+SalI. The resultingplasmid DNA is then cleaved with BamHI+HindIII restriction enzymes andthe LC/A-linker fragment inserted into a similarly cleaved vectorcontaining a unique multiple cloning site for BamHI, SalI, PstI, andHindIII such as the pMAL vector (NEB). The H_(N)/A DNA (SEQ ID NO:28) isthen cleaved with PstI+HindIII restriction enzymes and inserted into thesimilarly cleaved pMAL-LC/A-linker construct. The final constructcontains the LC/A-CPN(GS25)-H_(N)/A ORF (SEQ ID NO:72) for expression asa protein of the sequence illustrated in SEQ ID NO:73.

Variants of the LC/A-CPN-H_(N)/A fusion consisting of GS10, GS30 andHX27 are similarly created. Using the purification methodology describedin Example 26, fusion protein is purified from E. coli cell paste. FIG.12 illustrates the purified product obtained in the case ofLC/A-CPN(GS10)-H_(N)/A, LC/A-CPN(GS15)-H_(N)/A, LC/A-CPN(GS25)-H_(N)/A,LC/A-CPN(GS30)-H_(N)/A and LC/A-CPN(HX27)-H_(N)/A.

Example 30 Assessment of In Vitro Efficacy of an LC/A-Nociceptin-H_(N)/AFusion

Fusion protein prepared according to Examples 2 and 9 was assessed inthe eDRG neuronal cell model.

Assays for the inhibition of substance P release and cleavage of SNAP-25have been previously reported (Duggan et al., 2002, J. Biol. Chem., 277,34846-34852). Briefly, dorsal root ganglia neurons are harvested from15-day-old fetal Sprague-Dawley rats and dissociated cells plated onto24-well plates coated with Matrigel at a density of 1×10⁶ cells/well.One day post-plating the cells are treated with 10 μM cytosineβ-D-arabinofuranoside for 48 h. Cells are maintained in Dulbecco'sminimal essential medium supplemented with 5% heat-inactivated fetalbovine serum, 5 mM L-glutamine, 0.6% D-glucose, 2% B27 supplement, and100 ng/ml 2.5S mouse nerve growth factor. Cultures are maintained for 2weeks at 37° C. in 95% air/5% CO₂ before addition of test materials.

Release of substance P from eDRG is assessed by enzyme-linkedimmunosorbent assay. Briefly, eDRG cells are washed twice with lowpotassium-balanced salt solution (BSS: 5 mM KCl, 137 mM NaCl, 1.2 mMMgCl₂, 5 mM glucose, 0.44 mM KH₂PO₄, 20 mM HEPES, pH 7.4, 2 mM CaCl₂).Basal samples are obtained by incubating each well for 5 min. with 1 mlof low potassium BSS. After removal of this buffer, the cells arestimulated to release by incubation with 1 ml of high potassium buffer(BSS as above with modification to include 100 mM KCl isotonicallybalanced with NaCl) for 5 min. All samples are removed to tubes on iceprior to assay of substance P. Total cell lysates are prepared byaddition of 250 μl of 2 M acetic acid/0.1% trifluoroacetic acid to lysethe cells, centrifugal evaporation, and resuspension in 500 μl of assaybuffer. Diluted samples are assessed for substance P content. SubstanceP immunoreactivity is measured using Substance P Enzyme Immunoassay Kits(Cayman Chemical Company or R&D Systems) according to manufacturers'instructions. Substance P is expressed in pg/ml relative to a standardsubstance P curve run in parallel.

SDS-PAGE and Western blot analysis were performed using standardprotocols (Novex). SNAP-25 proteins were resolved on a 12% Tris/glycinepolyacrylamide gel (Novex) and subsequently transferred tonitrocellulose membrane. The membranes were probed with a monoclonalantibody (SMI-81) that recognises cleaved and intact SNAP-25. Specificbinding was visualised using peroxidase-conjugated secondary antibodiesand a chemiluminescent detection system. Cleavage of SNAP-25 wasquantified by scanning densitometry (Molecular Dynamics Personal SI,ImageQuant data analysis software). Percent SNAP-25 cleavage wascalculated according to the formula: (Cleaved SNAP-25/(Cleaved+IntactSNAP-25))×100.

Following exposure of eDRG neurons to an LC/A-nociceptin-H_(N)/A fusion(termed CPN-A), both inhibition of substance P release and cleavage ofSNAP-25 are observed (FIG. 13). After 24 h exposure to the fusion, 50%of maximal SNAP-25 cleavage is achieved by a fusion concentration of6.3±2.5 nM.

The effect of the fusion is also assessed at defined time pointsfollowing a 16 h exposure of eDRG to CPN-A. FIG. 14 illustrates theprolonged duration of action of the CPN-A fusion protein, withmeasurable activity still being observed at 28 days post exposure.

Example 31 Assessment of In Vitro Efficacy of an LC/A-NociceptinVariant-H_(N)/A Fusion

Fusion protein prepared according to Examples 8 and 9 was assessed inthe eDRG neuronal cell mode using the method described in Example 30.

Following exposure of eDRG neurons to an LC/A-nociceptin variant-H_(N)/Afusion (termed CPNv-A), both inhibition of substance P release andcleavage of SNAP-25 are observed. After 24 h exposure to the fusion, 50%of maximal SNAP-25 cleavage is achieved by a fusion concentration of1.4±0.4 nM (FIG. 15).

The effect of the fusion is also assessed at defined time pointsfollowing a 16 h exposure of eDRG to CPN-A. FIG. 16 illustrates theprolonged duration of action of the CPN-A fusion protein, withmeasurable activity still being observed at 24 days post exposure.

The binding capability of the CPNv-A fusion protein is also assessed incomparison to the CPN-A fusion. FIG. 17 illustrates the results of acompetition experiment to determine binding efficacy at the ORL-1receptor. CPNv-A is demonstrated to displace [3H]-nociceptin, therebyconfirming that access to the receptor is possible with the ligand inthe central presentation format.

Example 32 Preparation of an LC/A-Nociceptin Variant-H_(N)/A FusionProtein that is Activated by Treatment with Enterokinase

Following the methods used in Examples 1 and 2, the LC/A (SEQ ID NO:27)and H_(N)/A (SEQ ID NO:28) are created and inserted into the A serotypenociceptin variant linker arranged as BamHI-Sail-linker-enterokinaseprotease site-nociceptin variant-NheI-spacer-SpeI-PstI-XbaI-stopcodon-HindIII (SEQ ID NO:74). The final construct contains theLC-linker-nociceptin variant-spacer-H_(N) ORF sequences (SEQ ID NO:75)for expression as a protein of the sequence illustrated in SEQ ID NO:76.The fusion protein is termed CPNv(Ek)-A. FIG. 18 illustrates thepurification of CPNv(Ek)-A from E. coli following the methods used inExample 26 but using Enterokinase for activation at 0.00064 μg per 100μg of fusion protein.

Example 33 Assessment of In Vitro Efficacy of an LC/A-NociceptinVariant-H_(N)/A Fusion that has been Activated by Treatment withEnterokinase

The CPNv(Ek)-A prepared in Example 32 is obtained in a purified form andapplied to the eDRG cell model to assess cleavage of SNAP-25 (usingmethodology from Example 30). FIG. 19 illustrates the cleavage ofSNAP-25 following 24 h exposure of eDRG to CPNv(Ek)-A. The efficiency ofcleavage is observed to be similar to that achieved with the FactorXa-cleaved material, as recorded in Example 31.

Example 34 Preparation of an LC/C-Nociceptin Variant-H_(N)/C FusionProtein with a Factor Xa Activation Linker Derived from Serotype A

Following the methods used in Example 21, the LC/C (SEQ ID NO:31) andH_(N)/C (SEQ ID NO:32) are created and inserted into the A serotypenociceptin variant linker arranged as BamHI-Sail-linker-nociceptinvariant-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII II (SEQ ID NO:77).The final construct contains the LC-linker-nociceptinvariant-spacer-H_(N) ORF sequences (SEQ ID NO:78) for expression as aprotein of the sequence illustrated in SEQ ID NO:79. The fusion proteinis termed CPNv-C (act. A). FIG. 20 illustrates the purification ofCPNv-C (act. A) from E. coli following the methods used in Example 26.

Example 35 Assessment of In Vitro Efficacy of an LC/C-NociceptinVariant-H_(N)/C Fusion Protein

Following the methods used in Example 26, the CPNv-C (act. A) preparedin Example 34 is obtained in a purified form and applied to the eDRGcell model to assess cleavage of SNAP-25 (using methodology from Example30). After 24 h exposure to the fusion, 50% of maximal syntaxin cleavageis achieved by a fusion concentration of 3.1±2.0 nM. FIG. 21 illustratesthe cleavage of syntaxin following 24 h exposure of eDRG to CPNv-C (act.A).

Example 36 Assessment of In Vivo Efficacy of an LC/A-Nociceptin-HN/AFusion

The ability of an LC/A-nociceptin-H_(N)/A fusion (CPN/A) to inhibitacute capsaicin-induced mechanical allodynia is evaluated followingsubcutaneous intraplantar injection in the rat hind paw. Test animalsare evaluated for paw withdrawal frequency (PWF %) in response to a 10 gVon Frey filament stimulus series (10 stimuli x 3 trials) prior torecruitment into the study, after subcutaneous treatment with CPN/A butbefore capsaicin, and following capsaicin challenge post-injection ofCPN/A (average of responses at 15′ and 30′). Capsaicin challenge isachieved by injection of 10 μL of a 0.3% solution. Sample dilutions areprepared in 0.5% BSA/saline. FIG. 22 illustrates the reversal ofmechanical allodynia that is achieved by pre-treatment of the animalswith a range of concentrations of LC/A-nociceptin-HN/A fusion.

The ability of an LC/A-nociceptin-HN/A fusion (CPN/A) to inhibitstreptozotocin (STZ)-induced mechanical (tactile) allodynia in rats isevaluated. STZ-induced mechanical allodynia in rats is achieved byinjection of streptozotocin (i.p. or i.v.) which yields destruction ofpancreatic β-cells leading to loss of insulin production, withconcomitant metabolic stress (hyperglycemia and hyperlipidemia). Assuch, STZ induces Type I diabetes. In addition, STZ treatment leads toprogressive development of neuropathy, which serves as a model ofchronic pain with hyperalgesia and allodynia that may reflect signsobserved in diabetic humans (peripheral diabetic neuropathy).

Male Sprague-Dawley rats (250-300 g) are treated with 65 mg/kg STZ incitrate buffer (I.V.) and blood glucose and lipid are measured weekly todefine the readiness of the model. Paw Withdrawal Threshold (PWT) ismeasured in response to a Von Frey filament stimulus series over aperiod of time. Allodynia is said to be established when the PWT on twoconsecutive test dates (separated by 1 week) measures below 6 g on thescale. At this point, rats are randomized to either a saline group(negative efficacy control), gabapentin group (positive efficacycontrol) or a test group (CPN/A). Test materials (20-25 μl) are injectedsubcutaneously as a single injection (except gabapentin) and the PWT ismeasured at 1 day post-treatment and periodically thereafter over a2-week period. Gabapentin (30 mg/kg i.p.@3 ml/kg injection volume) isinjected daily, 2 hours prior to the start of PWT testing. FIG. 23illustrates the reversal of allodynia achieved by pre-treatment of theanimals with 750 ng of CPN/A. Data were obtained over a 2-week periodafter a single injection of CPN/A

Example 37 Assessment of In Vivo Efficacy of an LC/A-NociceptinVariant-H_(N)/A Fusion

The ability of an LC/A-nociceptin variant-H_(N)/A fusion (CPNv/A) toinhibit capsaicin-induced mechanical allodynia is evaluated followingsubcutaneous intraplantar injection in the rat hind paw. Test animalsare evaluated for paw withdrawal frequency (PWF %) in response to a 10 gVon Frey filament stimulus series (10 stimuli×3 trials) prior torecruitment into the study (Pre-Treat); after subcutaneous intraplantartreatment with CPNv/A but before capsaicin (Pre-CAP); and followingcapsaicin challenge post-injection of CPNv/A (average of responses at15′ and 30′; CAP). Capsaicin challenge is achieved by injection of 10 μLof a 0.3% solution. Sample dilutions are prepared in 0.5% BSA/saline.

FIG. 24 illustrates the reversal of allodynia that is achieved bypre-treatment of the animals with a range of concentrations ofLC/A-nociceptin variant-H_(N)/A fusion in comparison to the reversalachieved with the addition of LC/A-nociceptin-H_(N)/A fusion. These dataare expressed as a normalized paw withdrawal frequency differential, inwhich the difference between the peak response (post-capsaicin) and thebaseline response (pre-capsaicin) is expressed as a percentage. Withthis analysis, it can be seen that CPNv/A is more potent than CPN/Asince a lower dose of CPNv/A is required to achieve similar analgesiceffect to that seen with CPN/A.

Example 38 Preparation of an LC/A-Leu Enkephalin-H_(N)/A Fusion Protein

Due to the small, five-amino acid, size of the leu-enkephalin ligand theLC/A-leu enkephalin-H_(N)/A fusion is created by site directedmutagenesis [for example using Quickchange (Stratagene Inc.)] using theLC/A-nociceptin-H_(N)/A fusion (SEQ ID NO:39) as a template.Oligonucleotides are designed encoding the YGGFL leu-enkephalin peptide,ensuring standard E. coli codon usage is maintained and no additionalrestriction sites are incorporated, flanked by sequences complimentaryto the linker region of the LC/A-nociceptin-H_(N)/A fusion (SEQ IDNO:39) either side on the nociceptin section. The SDM product is checkedby sequencing and the final construct containing the LC-linker-leuenkephalin-spacer-H_(N) ORF (SEQ ID NO:80) for expression as a proteinof the sequence illustrated in SEQ ID NO:81. The fusion protein istermed CPLE-A. FIG. 25 illustrates the purification of CPLE-A from E.coli following the methods used in Example 26.

Example 39 Expression and Purification of an LC/A-Beta-Endorphin-H_(N)/AFusion Protein

Following the methods used in Example 26, and with theLC/A-beta-endorphin-H_(N)/A fusion protein (termed CPBE-A) created inExample 24, the CPBE-A is purified from E. coli. FIG. 26 illustrates thepurified protein as analysed by SDS-PAGE.

Example 40 Preparation of an LC/A-Nociceptin Mutant-H_(N)/A FusionProtein

Due to the single amino acid modification necessary to mutate thenociceptin sequence at position 1 from a Phe to a Tyr, theLC/A-nociceptin mutant-H_(N)/A fusion is created by site directedmutagenesis [for example using Quickchange (Stratagene Inc.)] using theLC/A-nociceptin-H_(N)/A fusion (SEQ ID NO:39) as a template.Oligonucleotides are designed encoding tyrosine at position 1 of thenociceptin sequence, ensuring standard E. coli codon usage is maintainedand no additional restriction sites are incorporated, flanked bysequences complimentary to the linker region of theLC/A-nociceptin-H_(N)/A fusion (SEQ ID NO:39) either side on thenociceptin section. The SDM product is checked by sequencing and thefinal construct containing the LC/A-nociceptin mutant-spacer-H_(N)/Afusion ORF (SEQ ID NO:82) for expression as a protein of the sequenceillustrated in SEQ ID NO:83. The fusion protein is termed CPOP-A. FIG.27 illustrates the purification of CPOP-A from E. coli following themethods used in Example 26.

Example 41 Preparation and Assessment of an LC/A-Nociceptin VariantMutant-H_(N)/A Fusion Protein

Due to the single amino acid modification necessary to mutate thenociceptin sequence at position 1 from a Phe to a Tyr, theLC/A-nociceptin variant mutant-H_(N)/A fusion is created by sitedirected mutagenesis [for example using Quickchange (Stratagene Inc.)]using the LC/A-nociceptin variant-H_(N)/A fusion (SEQ ID NO:51) as atemplate. Oligonucleotides are designed encoding tyrosine at position 1of the nociceptin sequence, ensuring standard E. coli codon usage ismaintained and no additional restriction sites are incorporated, flankedby sequences complimentary to the linker region of the LC/A-nociceptinvariant-H_(N)/A fusion (SEQ ID NO:51) either side on the nociceptinsection. The SDM product is checked by sequencing and the finalconstruct containing the LC/A-nociceptin mutant-spacer-H_(N)/A fusionORF (SEQ ID NO:84) for expression as a protein of the sequenceillustrated in SEQ ID NO:85. The fusion protein is termed CPOPv-A. FIG.28 illustrates the purification of CPOPv-A from E. coli following themethods used in Example 26.

Using methodology described in Example 30, CPOPv-A is assessed for itsability to cleave SNAP-25 in the eDRG cell model. FIG. 29 illustratesthat CPOPv-A is able to cleave SNAP-25 in the eDRG model, achievingcleavage of 50% of the maximal SNAP-25 after exposure of the cells toapproximately 5.9 nM fusion for 24 h.

Example 42 Preparation of an IgA Protease-Nociceptin Variant-H_(N)/AFusion Protein

The IgA protease amino acid sequence was obtained from freely availabledatabase sources such as GenBank (accession number P09790). Informationregarding the structure of the N. Gonorrhoeae IgA protease gene isavailable in the literature (Pohlner et al., Gene structure andextracellular secretion of Neisseria gonorrhoeae IgA protease, Nature,1987, 325(6103), 458-62). Using Backtranslation tool v2.0 (Entelechon),the DNA sequence encoding the IgA protease modified for E. coliexpression was determined. A BamHI recognition sequence was incorporatedat the 5′ end and a codon encoding a cysteine amino acid and SalIrecognition sequence were incorporated at the 3′ end of the IgA DNA. TheDNA sequence was screened using MapDraw, (DNASTAR Inc.) for restrictionenzyme cleavage sequences incorporated during the back translation. Anycleavage sequences that are found to be common to those required forcloning were removed manually from the proposed coding sequence isensuring common E. coli codon usage is maintained. E. coli codon usagewas assessed Graphical Codon Usage Analyser (Geneart), and the % GCcontent and codon usage ratio assessed by reference to published codonusage tables. This optimised DNA sequence (SEQ ID NO:86) containing theIgA open reading frame (ORF) is then commercially synthesized.

The IgA (SEQ ID NO:86) is inserted into the LC-linker-nociceptinvariant-spacer-H_(N) ORF (SEQ ID NO:51) using BamHI and SalI restrictionenzymes to replace the LC with the IgA protease DNA. The final constructcontains the IgA-linker-nociceptin variant-spacer-H_(N) ORF (SEQ IDNO:87) for expression as a protein of the sequence illustrated in SEQ IDNO:88.

Example 43 Preparation and Assessment of a Nociceptin TargetedEndopeptidase Fusion Protein with a Removable Histidine Purification Tag

DNA was prepared that encoded a Factor Xa removable his-tag (his6),although it is clear that alternative proteases site such asEnterokinase and alternative purification tags such as longer histidinetags are also possible. Using one of a variety of reverse translationsoftware tools [for example EditSeq best E. coli reverse translation(DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)], the DNAsequence encoding the Factor Xa removable his-tag region is determined.Restriction sites are then incorporated into the DNA sequence and can bearranged as NheI-linker-SpeI-PstI-H_(N)/A-XbaI-LEIEGRSGHHHHHHStopcodon-HindIII (SEQ ID NO:89). The DNA sequence is screened forrestriction sequence incorporated and any additional sequences areremoved manually from the remaining sequence ensuring common E.

colt codon usage is maintained. E. coli codon usage is assessed byreference to software programs such as Graphical Codon Usage Analyser(Geneart), and the % GC content and codon usage ratio assessed byreference to published codon usage tables (for example GenBank Release143, 13 Sep. 2004). This optimised DNA sequence is then commerciallysynthesized (for example by Entelechon, Geneart or Sigma-Genosys) and isprovided in the pCR 4 vector. In order to create CPNv-A-FXa-HT (SEQ IDNO:90, removable his-tag construct) the pCR 4 vector encoding theremovable his-tag is cleaved with NheI and HindIII. The NheI-HindIIIfragment is then inserted into the LC/A-CPNv-H_(N)/A vector (SEQ IDNO:51) that has also been cleaved by NheI and HindIII. The finalconstruct contains the LC/A-linker-nociceptinvariant-spacer-H_(N)-FXa-Histag-HindIII ORF sequences (SEQ ID NO:90) forexpression as a protein of the sequence illustrated in SEQ ID NO:91.FIG. 30 illustrates the purification of CPNv-A-FXa-HT from E. colifollowing the methods used in Example 26.

Example 44 Preparation of a Leu-Enkephalin Targeted Endopeptidase FusionProtein Containing a Translocation Domain Derived from Diphtheria Toxin

The DNA sequence is designed by back translation of the amino acidsequence of the translocation domain of the diphtheria toxin (obtainedfrom freely available database sources such as GenBank (accession number1×DTT) using one of a variety of reverse translation software tools [forexample EditSeq best E. coli reverse translation (DNASTAR Inc.), orBacktranslation tool v2.0 (Entelechon)]. Restriction sites are thenincorporated into the DNA sequence and can be arranged asNheI-Linker-SpeI-PstI-diphtheria translocation domain-XbaI-stopcodon-HindIII (SEQ ID NO:92). PstI/XbaI recognition sequences areincorporated at the 5′ and 3′ ends of the translocation domainrespectively of the sequence maintaining the correct reading frame. TheDNA sequence is screened (using software such as MapDraw, DNASTAR Inc.)for restriction enzyme cleavage sequences incorporated during the backtranslation. Any cleavage sequences that are found to be common to thoserequired by the cloning system are removed manually from the proposedcoding sequence ensuring common E. coli codon usage is maintained. E.coli codon usage is assessed by reference to software programs such asGraphical Codon Usage Analyser (Geneart), and the % GC content and codonusage ratio assessed by reference to published codon usage tables (forexample GenBank Release 143, 13 Sep. 2004). This optimised DNA sequencecontaining the diphtheria translocation domain is then commerciallysynthesized as NheI-Linker-SpeI-PstI-diphtheria translocationdomain-XbaI-stop codon-HindIII (for example by Entelechon, Geneart orSigma-Genosys) and is provided in the pCR 4 vector (Invitrogen). The pCR4 vector encoding the diphtheria translocation domain is cleaved withNheI and XbaI. The NheI-XbaI fragment is then inserted into theLC/A-CPLE-H_(N)/A vector (SEQ ID NO:80) that has also been cleaved byNheI and XbaI. The final construct contains theLC/A-leu-enkephalin-spacer-diphtheria translocation domain ORF sequences(SEQ ID NO:93) for expression as a protein of the sequence illustratedin SEQ ID NO:94.

Example 45 Preparation of a Nociceptin Variant Targeted EndopeptidaseFusion Protein Containing a LC Domain Derived from Tetanus Toxin

The DNA sequence is designed by back translation of the tetanus toxin LCamino acid sequence (obtained from freely available database sourcessuch as GenBank (accession number X04436) using one of a variety ofreverse translation software tools [for example EditSeq best E. colireverse translation (DNASTAR Inc.), or Backtranslation tool v2.0(Entelechon)]. BamHI/SalI recognition sequences are incorporated at the5′ and 3′ ends respectively of the sequence maintaining the correctreading frame (SEQ ID NO:95). The DNA sequence is screened (usingsoftware such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavagesequences incorporated during the back translation. Any cleavagesequences that are found to be common to those required by the cloningsystem are removed manually from the proposed coding sequence ensuringcommon E. coli codon usage is maintained. E. coli codon usage isassessed by reference to software programs such as Graphical Codon UsageAnalyser (Geneart), and the % GC content and codon usage ratio assessedby reference to published codon usage tables (for example GenBankRelease 143, 13 Sep. 2004). This optimised DNA sequence containing thetetanus toxin LC open reading frame (ORF) is then commerciallysynthesized (for example by Entelechon, Geneart or Sigma-Genosys) and isprovided in the pCR 4 vector (invitrogen). The pCR 4 vector encoding theTeNT LC is cleaved with BamHI and SalI. The BamHI-SalI fragment is theninserted into the LC/A-CPNv-H_(N)/A vector (SEQ ID NO:51) that has alsobeen cleaved by BamHI and SalI. The final construct contains the TeNTLC-linker-nociceptin variant-spacer-H_(N) ORF sequences (SEQ ID NO:96)for expression as a protein of the sequence illustrated in SEQ ID NO:97.

Example 46 Preparation of an LC/C-Nociceptin Variant-H_(N)/C FusionProtein with a Native Serotype C Linker that is Susceptible to Factor XaCleavage

Following the methods used in Example 21, the LC/C (SEQ ID NO:31) andH_(N)/C (SEQ ID NO:32) are created and inserted into the C serotypenociceptin variant linker arranged as BamHI-Sail-linker-nociceptinvariant-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII II (SEQ ID NO:98).The final construct contains the LC-linker-nociceptinvariant-spacer-H_(N) ORF sequences (SEQ ID NO:99) for expression as aprotein of the sequence illustrated in SEQ ID NO:100. The fusion proteinis termed CPNv-C (act. C).

Example 47 Construction of CHO-K1 OP2 Receptor Activation Assay andSNAP-25 Cleavage Assay Cell-Line Creation

CHO OP2 cell line was purchased from Perkin Elmer (ES-541-C, lot451-719-A). Cells were transfected with SNAP-25 DNA using Lipofectamine™2000 and incubated for 4 hours before media replacement. After 24 hours,cells were transferred to a T175 flask. 100 ug/ml Zeocin was added aftera further 24 hours to begin selection of SNAP-25 expressing cells, and 5ug/ml Blasticidin added to maintain selective pressure for the receptor.Cells were maintained in media containing selection agents for twoweeks, passaging cells every two to three days to maintain 30-70%confluence. Cells were then diluted in selective media to achieve 0.5cell per well in a 96 well microplate. After a few days, the plates wereexamined under a microscope, and those containing single colonies weremarked. Media in these wells was changed weekly. As cells becameconfluent in the wells, they were transferred to T25 flasks. When theyhad expanded sufficiently each clone was seeded to 24 wells of a 96 wellplate, plus a frozen stock vial created. LC/A-CPDY-H_(N)A fusion andLC/A-H_(N)A were applied to the cells for 24 hours, and then westernblots performed to detect SNAP-25 cleavage. Clones from which SNAP-25bands were strong and cleavage levels were high with fusion weremaintained for further investigation. Full dose curves were run onthese, and the clone (D30) with the highest differential betweenLC/A-CPDY-H_(N)A fusion and LC/A-H_(N)A cleavage levels was selected.

OP2 Receptor Activation Assay

The OP2 receptor activation measures the potency and intrinsic efficacyof ligands at OP2 receptor in transfected CHO-K1 cells by quantifyingthe reduction of forskolin-stimulated intracellular cAMP using aFRET-based cAMP (Perkin Elmer LANCE cAMP kit). After stimulation, afluorescently labelled cAMP tracer (Europium-streptavadin/biotin-cAMP)and fluorescently (Alexa) labelled anti-cAMP antibody are added to thecells in a lysis buffer. cAMP from the cells competes with the cAMPtracer for antibody binding sites. When read, a light pulse at 320 nmexcites the fluorescent portion (Europium) of the cAMP tracer. Theenergy emitted from the europium is is transferred to the Alexafluor-labelled antibodies bound to the tracer, generating a TR-FRETsignal at 665 nm (Time-resolved fluorescence resonance energy transferis based on the proximity of the donor label, europium, and the acceptorlabel, Alexa fluor, which have been brought together by a specificbinding reaction). Residual energy from the europium produces light at615 nm. In agonist treated cells there will be less cAMP to compete withthe tracer so a dose dependant increase in signal at 665 nm will beobserved compared with samples treated with forskolin alone. The signalat 665 nm signal is converted to cAMP concentration by interpolation toa cAMP standard curve which is included in each experiment.

Culture of Cells for Receptor Activation Assay:

Cells were seeded and cultured in T175 flasks containing Ham F12 withGlutamax, 10% Foetal bovine serum, 5 μml-1 Blasticidin and 100 μml-1Zeocin. The flasks were incubated at 37° C. in a humidified environmentcontaining 5% CO₂ until 60-80% confluent. On the day of harvest themedia was removed and the cells washed twice with 25 ml PBS. The cellswere removed from the flask by addition of 10 ml of Tryple Express, andincubation at 37° C. for 10 min followed by gentle tapping of the flask.The dislodged cells were transferred to a 50 ml centrifuge tube and theflask washed twice with 10 ml media which was added to the cellsuspension. The tube was centrifuged at 1300×g for 3 min and thesupernatant removed. Cells were gently re-suspended in 10 ml media (iffreezing cells) or assay buffer (if using ‘fresh’ cells in assay), and asample was removed for counting using a nucleocounter (ChemoMetec).Cells for use ‘fresh’ in an assay were diluted further in assay bufferto the appropriate concentration. Cells harvested for freezing werere-centrifuged (1300×g; 3 min), the supernatant removed and cellsre-suspended in Synth-a-freeze at 4° C. to 3×10⁶ cells/ml. Cryovialscontaining 1 ml suspension each were placed in a chilled Nalgene MrFrosty freezing container (−1° C./minute cooling rate), and leftovernight in a −80° C. freezer. The following day vials were transferredto the vapour phase of a liquid nitrogen storage tank.

Dilution of Test Materials and Cell Assay

Using Gilson pipettes and Sigmacoted or lo-bind tips, test materials andstandards were diluted to the appropriate concentrations in the wells ofthe first two columns of an eppendorf 500 μl deep-well lo-bind plate, inassay buffer containing 10 μM forskolin. The is chosen concentrations incolumns one and two were half a log unit apart. From these, serial 1:10dilutions were made across the plate (using an electronic eight channelpipette with sigmacote or lo-bind tips) until eleven concentrations athalf log intervals had been created. In the twelfth column, assay bufferonly was added as a ‘basal’. Using a 12 channel digital pipette, 10 μlof sample from the lo-bind plate was transferred to the optiplate 96well microplate.

To wells containing the standard curve, 10 ul of assay buffer was addedusing a multichannel digital pipette. To wells containing the testmaterials, 10 ul of cells in assay buffer at the appropriateconcentration were added. Plates were sealed and incubated for 120 minat room temperature, for the first hour on an IKA MTS 2/4 orbital shakerset to maximum speed.

Detection

LANCE Eu-W8044 labelled streptavidin (Eu-SA) and Biotin-cAMP (b-cAMP)were diluted in cAMP Detection Buffer (both from Perkin Elmer LANCE cAMPkit) to create sub-stocks, at dilution ratios of 1:17 and 1:5,respectively. The final detection mix was prepared by diluting from thetwo sub stocks into detection buffer at a ratio of 1:125. The mixturewas incubated for 15-30 min at room temperature before addition of 1:200Alexa Fluor® 647-anti cAMP Antibody (Alexa-Fluor Ab). After brieflyvortex mixing, 20 μl was immediately added to each well using a digitalmultichannel pipette. Microplate sealers were applied and platesincubated for 24 h at room temperature (for the first hour on an IKA MTS2/4 orbital shaker set to maximum speed). Plate sealers were removedprior to reading on the Envision.

FIGS. 36 and 37 show that dynorphin conjugates with LC/A-H_(N)/A,LC/B-H_(N)/B, LC/C—H_(N)/C and LC/D-H_(N)/D backbones active the OP2receptor.

CHO-K1 OP2 SNAP-25 Cleavage Assay

Cultures of cells were exposed to varying concentrations of fusionprotein for 24 hours. Cellular proteins were separated by SDS-PAGE andwestern blotted with anti-SNAP-25 antibody to facilitate assessment ofSNAP-25 cleavage. SNAP-25 cleavage calculated by densitometric analysis(Syngene).

Plating Cells

Prepare cells at 2×10e5 cells/ml and seed 125 μl per well of 96 wellplate. Use the following media: 500 ml Gibco Ham F12 with Glutamax(product code 31765068), 50 ml FBS, 5 ug/ml Blasticidin (250 μl aliquotfrom box in freezer, G13) (Calbiochem #203351, 10 ml at 10 mg/ml), 100ug/ml Zeocin (500 μl from box in freezer, G35). (Invitrogen from Fisher,1 g in 8×1.25 ml tubes at 100 mg/ml product code VXR25001). Allow cellsto grow for 24 hrs (37° C., 5% CO₂, humidified atmosphere).

Cell Treatment

Prepare dilutions of test protein for a dose range of each test proteins(make up double (2×) the desired final concentrations because 125 μlwill be applied directly onto 125 μl of media already in each well).Filter sterilize CHO KOR D30 feeding medium (20 ml syringe, 0.2 μmsyringe filter) to make the dilutions. Add the filtered medium into 5labelled bijoux's (7 ml tubes), 0.9 ml each using a Gilson pipette ormulti-stepper. Dilute the stock test protein to 2000 nM (working stocksolution 1) and 600 nM (working stock solution 2). Using a Gilsonpipette prepare 10-fold serial dilutions of each working stock, byadding 100 μl to the next concentration in the series. Pipette up anddown to mix thoroughly. Repeat to obtain 4 serial dilutions for solution1, and 3 serial dilutions for solution 2. A 0 nM control (filteredfeeding medium only) should also be prepared as a negative control foreach plate. Repeat the above for each test protein. In each experiment a‘standard’ batch of material must be included as control/referencematerial, this is unliganded LC/A-H_(N)/A.

Apply Diluted Sample to CHO KOR D30 Plates

Apply 125 μl of test sample (double concentration) per well. Each testsample should be applied to triplicate wells and each dose range shouldinclude a 0 nM control. Incubate for 24 hrs (37° C., 5% CO₂, humidifiedatmosphere).

Cell Lysis

Prepare fresh lysis buffer (20 mls per plate) with 25% (4×) NuPAGE LDSsample buffer, 65% dH₂O and 10% 1 M DTT. Remove medium from the CHO KORD30 plate by inverting over a waste receptacle. Drain the remainingmedia from each well using a fine-tipped pipette. Lyse the cells byadding 125 μl of lysis buffer per well using a multi-stepper pipette.After a minimum of 20 mins, remove the buffer from each well to a 1.5 mlmicrocentrifuge tube. Tubes must be numbered to allowing tracking of theCHO KOR treatments throughout the blotting procedure. A1-A3 down toH1-H3 numbered 1-24, A4-A6 down to H4-H6 numbered 25-48, A7-A9 down toH7-H93 numbered 49-72, A10-A12 down to H10-H12 numbered 73-96. Vortexeach sample and heat at 90° C. for 5-10 mins in a prewarmed heat block.Store at −20° C. or use on the same day on an SDS gel.

Gel Electrophoresis

If the sample has been stored o/n or longer, put in a heat blockprewarmed to 90° C. for 5-10 mins. Set up SDS page gels, use 1 gel per12 samples, prepare running buffer (1×, Invitrogen NuPAGE MOPS SDSRunning Buffer (20×) (NP0001))≈800 ml/gel tank. Add 500 μl of NuPAGEantioxidant to the upper buffer chamber. Load 15 ul samples onto gellanes from left to right as and load 2.5 ul of Invitrogen Magic MarkerXP and 5 ul Invitrogen See Blue Plus 2 pre-stained standard and 15 ul ofnon-treated control. It is important to maximize the resolution ofseparation during SDS_PAGE. This can be achieved by running 12% bis-trisgels at 200 V for 1 hour and 25 minutes (until the pink (17 kDa) markerreaches the bottom of the tank).

Western Blotting

Complete a Semi-dry transfer: using an Invitrogen iBlot (use iBlotProgramme 3 for 6 minutes). Put the nitrocellulose membranes inindividual small trays. Incubate the membranes with blocking buffersolution (5 g Marvel milk powder per 100 ml 0.1% PBS/Tween) at roomtemperature, on a rocker, for 1 hour. Apply primary antibody(Anti-SNAP-25 1:1000 dilution) and incubate the membranes with primaryantibody (diluted in blocking buffer) for 1 hour on a rocker at roomtemperature. Wash the membranes by rinsing 3 times with PBS/Tween(0.1%). Then apply the secondary (Anti-Rabbit-HRP conjugate diluted1:1000) and incubate the membranes with secondary antibody (diluted inblocking buffer) at room temperature, on a rocker, for 1 hour. Wash themembranes by rinsing 3 times with PBS/Tween (0.1%), leave membrane aminimum of 20 mins for the last wash. Detect the bound antibody usingSyngene: Drain blots of PBS/Tween, mix WestDura reagents 1:1 and add toblots for 5 minutes. Ensure enough solution is added to the membranes tocompletely cover them. Place membrane in Syngene tray, set up Syngenesoftware for 5 min expose time.

FIG. 34 clearly shows that LC/A-CPDY-H_(N)/A conjugates effectivelycleave SNAP-25.

Example 48 Construction and Activation of Dynorphin ConjugatesPreparation of a LC/A and H_(N)/A Backbone Clones

The following procedure creates the LC and H_(N) fragments for use asthe component backbone for multidomain fusion expression. This exampleis based on preparation of a serotype A based clone (SEQ ID NO:27 andSEQ ID NO:28), though the procedures and methods are equally applicableto the other serotypes [illustrated by the sequence listing for serotypeB (SEQ ID NO:29 and SEQ ID NO:30) and serotype C (SEQ ID NO:31 and SEQID NO:32)].

Preparation of Cloning and Expression Vectors

pCR 4 (Invitrogen) is the chosen standard cloning vector, selected dueto the lack of restriction sequences within the vector and adjacentsequencing primer sites for easy construct confirmation. The expressionvector is based on the pMAL (NEB) expression vector, which has thedesired restriction sequences within the multiple cloning site in thecorrect orientation for construct insertion (BamHI-SalI-PstI-HindIII). Afragment of the expression vector has been removed to create anon-mobilisable plasmid and a variety of different fusion tags have beeninserted to increase purification options.

Preparation of Protease (e.g. LC/A) InsertThe LC/A (SEQ ID NO:27) is created by one of two ways:

The DNA sequence is designed by back translation of the LC/A amino acidsequence [obtained from freely available database sources such asGenBank (accession number P10845) or Swissprot (accession locusBXA1_CLOBO) using one of a variety of reverse translation software tools(for example EditSeq best E. coli reverse translation (DNASTAR Inc.), orBacktranslation tool v2.0 (Entelechon)]. BamHI/SalI recognitionsequences are incorporated at the 5′ and 3′ ends respectively of thesequence, maintaining the correct reading frame. The DNA sequence isscreened (using software such as MapDraw, DNASTAR Inc.) for restrictionenzyme cleavage sequences incorporated during the back translation. Anycleavage sequences that are found to be is common to those required bythe cloning system are removed manually from the proposed codingsequence ensuring common E. coli codon usage is maintained. E. colicodon usage is assessed by reference to software programs such asGraphical Codon Usage Analyser (Geneart), and the % GC content and codonusage ratio assessed by reference to published codon usage tables (forexample GenBank Release 143, 13 Sep. 2004). This optimised DNA sequencecontaining the LC/A open reading frame (ORF) is then commerciallysynthesized (for example by Entelechon, Geneart or Sigma-Genosys) and isprovided in the pCR 4 vector.

The alternative method is to use PCR amplification from an existing DNAsequence with BamHI and SalI restriction enzyme sequences incorporatedinto the 5′ and 3′ PCR primers respectively. Complementaryoligonucleotide primers are chemically synthesised by a supplier (forexample MWG or Sigma-Genosys), so that each pair has the ability tohybridize to the opposite strands (3′ ends pointing “towards” eachother) flanking the stretch of Clostridium target DNA, oneoligonucleotide for each of the two DNA strands. To generate a PCRproduct the pair of short oligonucleotide primers specific for theClostridium DNA sequence are mixed with the Clostridium DNA template andother reaction components and placed in a machine (the ‘PCR machine’)that can change the incubation temperature of the reaction tubeautomatically, cycling between approximately 94° C. (for denaturation),55° C. (for oligonucleotide annealing), and 72° C. (for synthesis).Other reagents required for amplification of a PCR product include a DNApolymerase (such as Taq or Pfu polymerase), each of the four nucleotidedNTP building blocks of DNA in equimolar amounts (50-200 μM) and abuffer appropriate for the enzyme optimised for Mg²⁺ concentration(0.5-5 mM).

The amplification product is cloned into pCR 4 using either, TOPO TAcloning for Taq PCR products or Zero Blunt TOPO cloning for Pfu PCRproducts (both kits commercially available from Invitrogen). Theresultant clone is checked by sequencing. Any additional restrictionsequences which are not compatible with the cloning system are thenremoved using site directed mutagenesis [for example, using Quickchange(Stratagene Inc.)].

Preparation of Translocation (e.g. H_(N)) InsertThe H_(N)/A (SEQ ID NO:28) is created by one of two ways:

The DNA sequence is designed by back translation of the H_(N)/A aminoacid sequence [obtained from freely available database sources such asGenBank (accession number P10845) or Swissprot (accession locusBXA1_CLOBO)] using one of a variety of reverse translation softwaretools [for example EditSeq best E. coli reverse translation (DNASTARInc.), or Backtranslation tool v2.0 (Entelechon)]. A PstI restrictionsequence added to the N-terminus and XbaI-stop codon-HindIII to theC-terminus ensuring the correct reading frame is maintained. The DNAsequence is screened (using software such as MapDraw, DNASTAR Inc.) forrestriction enzyme cleavage sequences incorporated during the backtranslation. Any sequences that are found to be common to those requiredby the cloning system are removed manually from the proposed codingsequence ensuring common E. coli codon usage is maintained. E. colicodon usage is assessed by reference to software programs such asGraphical Codon Usage Analyser (Geneart), and the % GC content and codonusage ratio assessed by reference to published codon usage tables (forexample GenBank Release 143, 13 Sep. 2004). This optimised DNA sequenceis then commercially synthesized (for example by Entelechon, Geneart orSigma-Genosys) and is provided in the pCR 4 vector.

The alternative method is to use PCR amplification from an existing DNAsequence with PstI and XbaI-stop codon-HindIII restriction enzymesequences incorporated into the 5′ and 3′ PCR primers respectively. ThePCR amplification is performed as described above. The PCR product isinserted into pCR 4 vector and checked by sequencing. Any additionalrestriction sequences which are not compatible with the cloning systemare then removed using site directed mutagenesis [for example usingQuickchange (Stratagene Inc.)].

Preparation of Linker-Dynorphin-Spacer Insert

The LC-H_(N) linker can be designed from first principle, using theexisting sequence information for the linker as the template. Forexample, the serotype A linker (in this case defined as the inter-domainpolypeptide region that exists between the cysteines of the disulphidebridge between LC and H_(N)) is 23 amino acids long and has the sequenceVRGIITSKTKSLDKGYNKALNDL. Within this sequence, it is understood thatproteolytic is activation in nature leads to an H_(N) domain that has anN-terminus of the sequence ALNDL. This sequence information is freelyavailable from available database sources such as GenBank (accessionnumber P10845) or Swissprot (accession locus BXA1_CLOBO). Into thislinker an enterokinase site, dynorphin and spacer are incorporated; andusing one of a variety of reverse translation software tools [forexample EditSeq best E. coli reverse translation (DNASTAR Inc.), orBacktranslation tool v2.0 (Entelechon)], the DNA sequence encoding thelinker-ligand-spacer region is determined. Restriction sites are thenincorporated into the DNA sequence and can be arranged asBamHI-SalI-linker-proteasesite-dynorphin-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII. It isimportant to ensure the correct reading frame is maintained for thespacer, dynorphin and restriction sequences and that the XbaI sequenceis not preceded by the bases, TC, which would result on DAM methylation.The DNA sequence is screened for restriction sequence incorporation, andany additional sequences are removed manually from the remainingsequence ensuring common E. coli codon usage is maintained. E. colicodon usage is assessed by reference to software programs such asGraphical Codon Usage Analyser (Geneart), and the % GC content and codonusage ratio assessed by reference to published codon usage tables (forexample, GenBank Release 143, 13 Sep. 2004). This optimised DNA sequenceis then commercially synthesized (for example by Entelechon, Geneart orSigma-Genosys) and is provided in the pCR 4 vector.

Preparation of the LC/A-Dynorphin-H_(N)/A Fusion

In order to create the LC-linker-dynorphin-spacer-H_(N) construct (SEQID NO:102), the pCR 4 vector encoding the linker-dynorphin-spacer iscleaved with BamHI+SalI restriction enzymes. This cleaved vector thenserves as the recipient vector for insertion and ligation of the LC/ADNA (SEQ ID NO:27) cleaved with BamHI+SalI. The resulting plasmid DNA isthen cleaved with PstI+XbaI restriction enzymes and serves as therecipient vector for the insertion and ligation of the H_(N)/A DNA (SEQID NO:28) cleaved with PstI+XbaI. The final construct contains theLC-linker-dynorphin-spacer-H_(N) ORF (SEQ ID NO:102) for transfer intoexpression vectors for expression to result in a fusion protein of thesequence illustrated in SEQ ID NO:103.

Examples 49 Preparation and Purification of an LC/A-Dynorphin-H_(N)/AFusion Protein Family with Variable Spacer Length

Using the same strategy as employed in Example 48, a range of DNAlinkers were prepared that encoded dynorphin and variable spacercontent. Using one of a variety of reverse translation software tools[for example EditSeq best E. coli reverse translation (DNASTAR Inc.), orBacktranslation tool v2.0 (Entelechon)], the DNA sequence encoding thelinker-ligand-spacer region is determined. Restriction sites are thenincorporated into the DNA sequence and can be arranged asBamHI-Sail-linker-proteasesite-dynorphin-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII. It isimportant to ensure the correct reading frame is maintained for thespacer, dynorphin and restriction sequences and that the XbaI sequenceis not preceded by the bases, TC which would result on DAM methylation.The DNA sequence is screened for restriction sequence incorporation andany additional sequences are removed manually from the remainingsequence ensuring common E. coli codon usage is maintained. E. colicodon usage is assessed by reference to software programs such asGraphical Codon Usage Analyser (Geneart), and the % GC content and codonusage ratio assessed by reference to published codon usage tables (forexample GenBank Release 143, 13 Sep. 2004). This optimised DNA sequenceis then commercially synthesized (for example by Entelechon, Geneart orSigma-Genosys) and is provided in the pCR 4 vector.

The spacers that were created included are shown in Table 2 above (seeExample 29).

By way of example, in order to create the LC/A-CPDY(GS25)-H_(N)/A fusionconstruct (SEQ ID NO:106), the pCR 4 vector encoding the linker iscleaved with BamHI+SalI restriction enzymes. This cleaved vector thenserves as the recipient vector for insertion and ligation of the LC/ADNA (SEQ ID NO:27) also cleaved with BamHI+SalI. The resulting plasmidDNA is then cleaved with BamHI+HindIII restriction enzymes and theLC/A-linker fragment inserted into a similarly cleaved vector containinga unique multiple cloning site for BamHI, SalI, PstI, and HindIII suchas the pMAL vector (NEB). The H_(N)/A DNA (SEQ ID NO:28) is then cleavedwith PstI+HindIII restriction enzymes and inserted into the similarlycleaved pMAL-LC/A-linker construct. The final construct contains theLC/A-CPDY(GS25)-H_(N)/A ORF for expression as a protein of the sequenceillustrated in SEQ ID NO:106.

Example 50 Purification Method for LC/A-Dynorphin-H_(N)/A Fusion Protein

Defrost falcon tube containing 25 ml 50 mM HEPES pH 7.2, 200 mM NaCl andapproximately 10 g of E. coli BL21 cell paste. Make the thawed cellpaste up to 80 ml with 50 mM HEPES pH 7.2, 200 mM NaCl and sonicate onice 30 seconds on, 30 seconds off for 10 cycles at a power of 22 micronsensuring the sample remains cool. Spin the lysed cells at 18 000 rpm, 4°C. for 30 minutes. Load the supernatant onto a 0.1 M NiSO₄ chargedChelating column (20-30 ml column is sufficient) equilibrated with 50 mMHEPES pH 7.2, 200 mM NaCl. Using a step gradient of 10 and 40 mMimidazol, wash away the non-specific bound protein and elute the fusionprotein with 100 mM imidazol. Dialyse the eluted fusion protein against5 L of 50 mM HEPES pH 7.2, 200 mM NaCl at 4° C. overnight and measurethe OD of the dialysed fusion protein. Add 3.2 μl of enterokinase (2μg/ml) per 1 mg fusion protein and Incubate at 25° C. static overnight.Load onto a 0.1 M NiSO₄ charged Chelating column (20-30 ml column issufficient) equilibrated with 50 mM HEPES pH 7.2, 200 mM NaCl. Washcolumn to baseline with 50 mM HEPES pH 7.2, 200 mM NaCl. Using a stepgradient of 10 and 40 mM imidazol, wash away the non-specific boundprotein and elute the fusion protein with 100 mM imidazol. Dialyse theeluted fusion protein against 5 L of 50 mM HEPES pH 7.2, 200 mM NaCl at4° C. overnight and concentrate the fusion to about 2 mg/ml, aliquotsample and freeze at −20° C. Test purified protein using OD, BCA, purityanalysis and SNAP-25 assessments.

Example 51 Preparation of a LC/C-Dynorphin-H_(N)/C Fusion Protein with aSerotype A Activation Sequence

Following the methods used in Examples 18 and 19, the LC/C (SEQ IDNO:31) and H_(N)/C (SEQ ID NO:32) are created and inserted into the Aserotype linker arranged as BamHI-SalI-linker-proteasesite-dynorphin-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII. The finalconstruct contains the LC-linker-dynorphin-spacer-H_(N) ORF forexpression as a protein of the sequence illustrated in SEQ ID NO:107.

Example 52 Preparation of an IgA Protease-Dynorphin Variant-H_(N)/AFusion Protein

The IgA protease amino acid sequence was obtained from freely availabledatabase sources such as GenBank (accession number P09790). Informationregarding the structure of the N. Gonorrhoeae IgA protease gene isavailable in the literature (Pohlner et al., Gene structure andextracellular secretion of Neisseria gonorrhoeae IgA protease, Nature,1987, 325(6103), 458-62). Using Backtranslation tool v2.0 (Entelechon),the DNA sequence encoding the IgA protease modified for E. coliexpression was determined. A BamHI recognition sequence was incorporatedat the 5′ end and a codon encoding a cysteine amino acid and SalIrecognition sequence were incorporated at the 3′ end of the IgA DNA. TheDNA sequence was screened using MapDraw, (DNASTAR Inc.) for restrictionenzyme cleavage sequences incorporated during the back translation. Anycleavage sequences that are found to be common to those required forcloning were removed manually from the proposed coding sequence ensuringcommon E. coli codon usage is maintained. E. coli codon usage wasassessed Graphical Codon Usage Analyser (Geneart), and the % GC contentand codon usage ratio assessed by reference to published codon usagetables. This optimised DNA sequence (SEQ ID NO:86) containing the IgAopen reading frame (ORF) is then commercially synthesized.

The IgA (SEQ ID NO:86) is inserted into theLC-linker-dynorphin-spacer-H_(N) ORF (SEQ ID NO:102) using BamHI andSalI restriction enzymes to replace the LC with the IgA protease DNA.The final construct contains the IgA-linker-dynorphin-spacer-H_(N) ORFfor expression as a protein of the sequence illustrated in SEQ IDNO:108.

Example 53 Preparation of a Dynorphin Targeted Endopeptidase FusionProtein Containing a LC Domain Derived from Tetanus Toxin

The DNA sequence is designed by back translation of the tetanus toxin LCamino acid sequence (obtained from freely available database sourcessuch as GenBank (accession number X04436) using one of a variety ofreverse translation software tools [for example EditSeq best E. colireverse translation (DNASTAR Inc.), or Backtranslation tool v2.0(Entelechon)]. BamHI/SalI recognition sequences are incorporated at the5′ and 3′ ends respectively of the sequence maintaining the correctreading frame (SEQ ID NO:95). The DNA sequence is screened (usingsoftware such is as MapDraw, DNASTAR Inc.) for restriction enzymecleavage sequences incorporated during the back translation. Anycleavage sequences that are found to be common to those required by thecloning system are removed manually from the proposed coding sequenceensuring common E. coli codon usage is maintained. E. coli codon usageis assessed by reference to software programs such as Graphical CodonUsage Analyser (Geneart), and the % GC content and codon usage ratioassessed by reference to published codon usage tables (for exampleGenBank Release 143, 13 Sep. 2004). This optimised DNA sequencecontaining the tetanus toxin LC open reading frame (ORF) is thencommercially synthesized (for example by Entelechon, Geneart orSigma-Genosys) and is provided in the pCR 4 vector (invitrogen). The pCR4 vector encoding the TeNT LC is cleaved with BamHI and SalI. TheBamHI-SalI fragment is then inserted into the LC/A-dynorphin-H_(N)/Avector (SEQ ID NO:102) that has also been cleaved by BamHI and SalI. Thefinal construct contains the TeNT LC-linker-dynorphin-spacer-H_(N) ORFsequences for expression as a protein of the sequence illustrated in SEQID NO:109.

Example 54 Preparation and Purification of an LC/A-Dynorphin-H_(N)/AFusion Protein Family with Variable Dynorphin Ligands

Using the same strategy as employed in Example 48, a range of DNADynorphin ligands were prepared that encoded various dynorphin ligands.Using one of a variety of reverse translation software tools [forexample EditSeq best E. coli reverse translation

(DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)], the DNAsequence encoding the linker-ligand-spacer region is determined.Restriction sites are then incorporated into the DNA sequence and can bearranged as BamHI-SalI-linker-protease site-dynorphinligand-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII. It is important toensure the correct reading frame is maintained for the spacer, dynorphinligand and restriction sequences and that the XbaI sequence is notpreceded by the bases, TC which would result on DAM methylation. The DNAsequence is screened for restriction sequence incorporation and anyadditional sequences are removed manually from the remaining sequenceensuring common E. coli codon usage is maintained. E. coli codon usageis assessed by reference to software programs such as Graphical CodonUsage Analyser (Geneart), and the % GC content and codon usage ratioassessed by reference to published codon usage tables (for exampleGenBank Release 143, 13 Sep. 2004). This optimised DNA sequence is thencommercially synthesized (for example by Entelechon, Geneart orSigma-Genosys) and is provided in the pCR 4 vector.

Alternatively, the dynorphin ligand was created by performingsite-directed mutagenesis on the DNA sequence of LC/A-CPDY-HN/A fusion(SEQ ID NO102).

The ligands that were created included:

SEQ ID NO of Dynorphin Protein sequence of the Dynorphin Ligandthe Dynorphin ligands ligand CPDY1-13 YGGFLRRIRPKLK 113 CPDY(D15A)YGGFLRRIRPKLKWANQ 114 CPDY YGGFLRRIRPKLK 118 (18RP10R)1-13 CPDY(I8RP1-YGGFLRRRRRKLKWANQ 117 RD15A) CPDNv9 YGGFLGARKSARKRKNQ 119

By way of example, in order to create the LC/A-CPDY(D15A)-GS20-H_(N)/Afusion construct (SEQ ID NO114), the pCR 4 vector encoding the fusionprotein (SEQ ID NO102) serves as a template for site-directedmutagenesis to mutate the aspartic acid residue at position 15 withinthe dynorphin ligand to alanine. A forward and reverse primer wasdesigned and synthesised that were complementary to the template DNAapart but encoded a mismatch to incorporate the required mutation. 125ng primers, 1 μl dNTPs, 5-50 ng template DNA, 5 μl of 10× reactionbuffer and 1μ Pfu polymerase (2.5 U/μl) were added to a 50 μl reactionmixture. The PCR reaction was as follows: 95° C. for 2 min, then 24cycles of 95° C. for 1 min, 55° C. annealing for 1 min, 68° C. finalextension for 8 min, then a 4° C. hold. The DNA product was thentransformed into TOP10 cells and the plasmid DNA from the resultingcolonies was then purified and sequenced to confirm that the dynorphinligand had been mutated to create a DNA construct that will give the ORFLC/A-CPDY(D15A)-GS20-H_(N)/A (SEQ ID NO 114).

Purification of Purification of an LC/A-Dynorphin-H_(N)/A Fusion ProteinFamily with Variable Dynorphin Ligands

Defrost falcon tube containing 25 ml 50 mM HEPES pH 7.2, 200 mM NaCl andapproximately 10 g of E. coli BL21 cell paste. Make the thawed cellpaste up to 80 ml with 50 mM HEPES pH 7.2, 200 mM NaCl and The cellpaste was then homogenised at 20,000 psi by a Constant SystemHomogeniser. Spin the lysed cells at 18 000 rpm, 4° C. for 30 minutes.Load the supernatant onto a 0.1 M NiSO₄ charged Chelating column (20-30ml column is sufficient) equilibrated with 50 mM HEPES pH 7.2, 200 mMNaCl. Using a step gradient of 10 and 40 mM imidazol, wash away thenon-specific bound protein and elute the fusion protein with 100 mMimidazol. Dialyse the eluted fusion protein against 5 L of 50 mM HEPESpH 7.2, 200 mM NaCl at 4° C. overnight and measure the OD of thedialysed fusion protein. Add 3.2 μl of enterokinase (2 μg/ml) per 1 mgfusion protein and Incubate at 25° C. static overnight. Activatedsamples were then subjected to hydrophobic interaction chromatography(HIC purification). Solid ammonium sulphate was slowly added by spatulato the activated fusion protein and stirred by a magnetic flea at roomtemperature to dissolve the solid. More ammonium sulphate was added oncethe previous addition had been dissolved and this was repeated until theconcentration reached 1 M. Load onto a Phenyl sepharose 6 fast flow(20-30 ml column is sufficient) equilibrated with 50 mM HEPES pH 7.2, 1M ammonium sulphate. The column was then washed with 50 mM hepes pH 7.2,1 M ammonium sulphate. Bound protein was eluted by reducing the ammoniumsulphate concentration to 0.7 M, 0.5 M, 0.3 M and 0 M and collected in10 ml fractions. Dialyse the eluted fusion protein against 10 L of 50 mMHEPES pH 7.2, 200 mM NaCl at 4° C. overnight and concentrate the fusionto about 2 mg/ml, aliquot sample and freeze at −20° C. Test purifiedprotein using OD, BCA, purity analysis and SNAP-25 assessments.

Example 55 Preparation and Purification of an LC/A-Dynorphin1-13-H_(N)/A(GS30) and LC/A-Dynorphin1(D15A)-H_(N)/A (GS30) Fusion Protein

Using the constructs described in Examples 48 and Example 54,LC/A-dynorphin1-13-H_(N)/A (GS30) and LC/A-dynorphin1(D15A)-H_(N)/A(GS30) DNA encoding fusion proteins (SEQ ID NOs 116 and 115) werecreated by sub-cloning experiments. By way of an example, the DNAencoding LC/A-dynorphin1(D15A)-HN/A fusion protein (SEQ ID NO 114) wasdigested with the restriction enzymes NheI and HindIII to remove theGS20 spacer and heavy chain. DNA encoding LC/A-dynorphin-HN/A (GS30)fusion protein (SEQ ID NO 110) was also digested with NheI and HindIIIto produce a GS30-H_(N)/A fragment. Vector backbone and fragment DNA wasseparated by running on a 1% agroase gel at 150 volts for 1 hour beforethe DNA was purified with a gel extraction kit. The GS30-H_(N)/Afragment was then ligated into the vector backbone of digestedLC/A-dynorphin1(D15A)-H_(N)/A fusion protein (SEQ ID NO 114) to produceLC/A-dynorphin1(D15A)-H_(N)/A (GS30) fusion protein. Typical ligationreactions were set up as follows: 2 μl Vector DNA, 14 μl Insert DNA, 2μl 10× T4 ligase buffer, 2 μl T4 ligase. Ligation reactions were leftovernight at 16° C.

Purification of LC/A-Dynorphin1-13-H_(N)/A (GS30) andLC/A-dynorphin1(D15A)-H_(N)/A (GS30) Fusion Protein

The fusion proteins were purified as described in Example 54.

Example 56 Preparation of a LC/B-Dynorphin-H_(N)/B Fusion Protein with aSerotype A Activation Sequence

Following the methods used in Examples 18 and 19, the LC/B (SEQ ID NO29)and H_(N)/B (SEQ ID NO30) were created and inserted into the A serotypelinker arranged as BamHI-SalI-linker-proteasesite-dynorphin-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII. The finalconstruct contained the LB-linker-dynorphin-spacer-H_(N)/B ORF forexpression as a protein of the sequence illustrated in SEQ ID NO112.

Example 57 Construction of CHO-K1 MrgX1 Receptor Activation Ca2+Fluorimetry Assay Cell-Line Culture

CHO-K1 MrgX1 Cell Line cells were purchased from Perkin Elmer (PerkinElmer ES-740-A). Cell culture media used was F-12 HAMS+Glutamax with 10%Foetal bovine serum and 800 μg/ml Geneticin. Cells were grown incontinuous culture in T500 flasks. Subconfluent cultures (70-80%)confluent were split at a ratio 1:40 every 3-4 days. Then the culturemedium was removed from the cells and the cells washed twice with 30 mLPBS. Cells were then removed from the flask by addition of 30 ml ofPBS-based enzyme free dissociation buffer, and incubated at 37 C for 5min followed by gentle tapping of the flasks to dislodge cells. Added10-20 mL of culture media to the flasks and transferred the remainingcells to a 50 ml tube. Washed the flask with 10-20 ml culture media andadded this to the cell suspension. Cells were then centrifuged at 1300rpm for 3 min in a Heraeus megafuge 1.0 to pellet cells before removingthe supernatant and resuspending the cell pellet in culture media. Cellswere then diluted further with culture media to achieve a split ratio of1:40 and transferred appropriate volume of cells to a T500 flask. Thenumber of cells present were counted using the Nucleocounter. Cells werethen pelleted by centrifugation at 1,300 rpm for 3 min in a Heraeusmegafuge 1.0 and the pellet re-suspend in Bambanker freezing medium toachieve a concentration of 3×106 cells·mL−1. After aliquoting the cellsuspension into 1.8 mL Nunc cryovials, the vials were transferred to acryo vial rack and store at −80° C. overnight before transferring thevials to the short-term liquid nitrogen store. Several cell passagenumbers were frozen in liquid nitrogen for use in the assay. Maximumpassage number to be used in the assay is Px+16.

Resurrection and Plating CHO-K1-MrgX1 Cells

This assay was optimized for the use of frozen cells. Plated cells ˜16 hbefore the assay performed. Removed cell vials from liquid nitrogen andthawed quickly by placing vials in a 37° C. water bath. Transferredcells to a centrifuge tube containing 10 ml growth media for each vialbeing resurrected. Pelleted the cells by centrifuging at 1,300 rpm for 3min in a Heraeus megafuge 1.0. Then the cell pellet was re-suspend in 2mL media/vial and the number of viable cells in the suspension countedusing a Nucleocounter before adding media to the cell suspension toachieve a cell concentration of 1×106 cells·mL−1. Then the cells wereplated in a Corning black walled, clear-bottom, half-area plate at adensity of 20,000 viable cells·well−1 by using a Rainin E8-300multi-channel pipette to firstly add 30 μL media to each well followedby 20 μL·well-1 of the 1×106 cells·mL−1 cell suspension. Cells wereplated 16-18 h prior to the beginning of the assay and the platesmaintain under normal growth conditions in a 37° C. 5% CO₂ incubator.

HBSS Assay Buffer Preparation

Added 1.26 mmol·L⁻¹ CaCl₂ (630 μL of 1 mol·L⁻¹ CaCl2), 0.49 mmol·L⁻¹MgCl₂ (245 μL of 1 mol·L⁻¹ MgCl₂), 0.4 mmol·L⁻¹ MgSO₄ (203 μL of 1mol·L−1 MgSO₄), 20 mmol·L⁻¹ HEPES (10 mL of 1 mol·L⁻¹ HEPES) to 500 mLHBSS. Adjust the pH of the HBSS buffer to pH 7.4 at room temperatureusing NaOH. Filter sterilise the HBSS buffer in a sterile hood. On theday of the assay, prepared a fresh stock of Probenecid at 500 mmol·L−1by dissolving 710 mg of probenecid (MW: 285.36) in 5 mL of 1 mol·L−1NaOH. Assay buffer was made by adding the appropriate volume of 500mmol·L⁻¹ probenecid to HBSS buffer required for the assay plate (finalconcentration, 5 mmol·L⁻¹) and the ligand dilution series (2.5 mmol·L⁻¹final concentration). To the 5 mmol·L⁻¹ probenecid/HBSS assay bufferadded a volume of 10% BSA that gives a final concentration of 0.02° A)BSA. Used the same solution of 5 mmol·L⁻¹ probenecid/0.02% BSA assaybuffer to dilute 1 in 2 in HBSS to make a 2.5 mmol·L−1 probenecid/0.01%BSA solution for ligand dilution.

Calcium-3 Dye Preparation

Added 10 mL HBSS buffer to a bottle of desiccated Ca²⁺-4 dye. Vortexedhard and transfer to a 100 mL container. Repeated this 9 times so that atotal of 100 mL HBSS buffer had been added to the Ca²⁺⁻4 dye. Aliquotedthe dye into 10 mL aliquots and stored at −20° C. When using a frozenaliquot, the vial was removed from −20° C. freezer and warmed in a 37°C. waterbath. After thawing the necessary number of vials of Ca²⁺−4 dye,it was diluted 1:2 with HBSS buffer to attain 0.5×Ca²⁺-4 dye.

Dilution of Test Fusions

Prepared the source plate, containing ligand or fusion, prior to loadingcells and beginning their incubation. All reference ligand concentrationranges were achieved by serial dilution in half-log 10 increments usingSigmacoted tips. The reference compound BAM (8-22) was included in everyassay at a concentration range of 5×10⁻⁶M (5 μM) to 5×10−11 M (50 pM)(final assay concentration will be 5× lower) plus basal (1×10−14).Fusions to be tested were included in every assay at a concentrationrange of 5×10−6 M to 5×10−9 M (final assay concentration will be 5×lower).

Prepared an intermediate 50 μmol·L−1 stock of BAM (8-22) by 1 in 10dilution in HBSS assay buffer (2.5 mmol·L−1 probenecid; 0.01% BSA HBSSassay buffer) of 500 μmol·L-1 stock using lo-bind Eppendorf tubes andGilson P20 and P100 pipettes. Then, transfer 50 μL of the 50 μmol·L−1intermediate stock to the first well of a 0.5 mL lo-bind 96-well platecontaining 450 μL of HBSS buffer and performed the ligand dilutionseries creating 1:10 dilution series going down the plate.

The assay was optimized for the source plate layout in rows, thereforesource the dilution series must be split into triplicates (minimum of 50μL to allow FlexStation3 to transfer 25 μL) in a separate 0.5 mL lo-bind96-well plate (×2 compounds per plate). This can be transferred directlyto the FlexStation3 for ligand transfer using Sigmacoted FlexStation3tips (Molecular Devices).

Dye Lading of Cells

Removed culture media from the half area 96-well plates containingcells, incubated overnight using a Rainin L50 pipette, taking care notto disrupt cells. Add 50 μL of assay buffer followed by 50 μL of0.5×Ca2+ dye using an electronic multichannel pipette E8-300. Incubatecells at 37° C. in 5% CO2 for 120 min.

FLEXSTATION3 Readings

The human mas-related G-protein coupled receptor member X1 belongs tothe family of orphan G protein-coupled receptors. Predominantly coupledthrough Gαq/11, receptor activation by an agonist causes Gag proteinactivation resulting in Ca²⁺ release from intracellular stores that ismediated by the target enzyme phospholipase Cβ. The transient increasein intracellular Ca²⁺ requires a real-time (RT), simultaneousinject-and-read system to measure Ca²⁺ flux. The FlexStation3 microplatereader with integrated fluid transfer is used in this assay for thispurpose. CHO cells that express the recombinant human MrgX1 receptor areincubated with the proprietary FLIPR-Calcium-4 masking dye thatminimises background signal from extracellular Ca²⁺ and makes washingcells unnecessary. The Ca²⁺-4 dye forms a complex with Ca²⁺ whichfluoresces at 525 nm following excitation at 485 nm allowingsignal-detection. An inhibitor of cell membrane anion exchanger,probenecid, is included in the assay buffer to prevent outward transportor sequestration of dye molecules. Following incubation with the dye,the cell plate is loaded onto to the FlexStation3 which transfersligands (reference agonist or fusions) from a source plate into themicroplate wells containing cells. The FlexStation 3 measures thefluorescent-emission from the Calcium-4 dye and readouts are formed ascalcium traces displaying the magnitude of calcium flux as a result ofMrgX1 receptor activation.

Example 58 Construction and Activation of BAM Fusion Proteins

To construct fusions that contain BAM1-22 (SEQ ID NO120) and BAMS-22(SEQ ID NO121) the preparation of a LC/A and H_(N)/A backbone clones andpreparation of cloning and expression vectors are identical as thosedescribed in Example 48.

Preparation of Linker-BAM-Spacer Insert

The LC-H_(N) linker can be designed from first principle, using theexisting sequence information for the linker as the template. Forexample, the serotype A linker (in this case defined as the inter-domainpolypeptide region that exists between the cysteines of the disulphidebridge between LC and H_(N)) is 23 amino acids long and has the sequenceVRGIITSKTKSLDKGYNKALNDL. Within this sequence, it is understood thatproteolytic activation in nature leads to an H_(N) domain that has anN-terminus of the sequence ALNDL. This sequence information is freelyavailable from available database sources such as GenBank (accessionnumber P10845) or Swissprot (accession locus BXA1_CLOBO). Into thislinker an enterokinase site, BAM ligand (SEQ ID NO 120 or SEQ ID NO 121)and spacer are incorporated; and using one of a variety of reversetranslation software tools [for example EditSeq best E. coli reversetranslation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)],the DNA sequence encoding the linker-ligand-spacer region is determined.Restriction sites are then incorporated into the DNA sequence and can bearranged as BamHI-Sail-linker-protease site-BAM(1-22) or BAM(8-22)ligand-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII. It is important toensure the correct reading frame is maintained for the spacer, dynorphinand restriction sequences and that the XbaI sequence is not preceded bythe bases, TC, which would result on DAM methylation. The DNA sequenceis screened for restriction sequence incorporation, and any additionalsequences are removed manually from the remaining sequence ensuringcommon E. coli codon usage is maintained. E. coli codon usage isassessed by reference to software programs such as Graphical Codon UsageAnalyser (Geneart), and the % GC content and codon usage ratio assessedby reference to published codon usage tables (for example, GenBankRelease 143, 13 Sep. 2004). This optimised DNA sequence is thencommercially synthesized (for example by Entelechon, Geneart orSigma-Genosys) and is provided in the pCR 4 vector.

Preparation of the LC/A-BAM(1-22)-H_(N)/A Fusion

In order to create the LC-linker-BAM(1-22)-spacer-H_(N)/A construct (SEQID NO122), the pCR 4 vector encoding the linker-BAM(1-22)-spacer insertis cleaved with BamHI+SalI restriction enzymes. This cleaved vector thenserves as the recipient vector for insertion and ligation of the LC/ADNA (SEQ ID NO27) cleaved with BamHI+SalI. The resulting plasmid DNA isthen cleaved with PstI+XbaI restriction enzymes and serves as therecipient vector for the insertion and ligation of the H_(N)/A DNA (SEQID NO28) cleaved with PstI+XbaI. The final construct contains theLC-linker-BAM(1-22)-spacer-H_(N)/A ORF (SEQ ID NO122) for transfer intoexpression vectors for expression to result in a fusion protein of thesequence illustrated in SEQ ID NO123.

Preparation of the LC/A-BAM(8-22)-H_(N)/A Fusion

In order to create the LC-linker-BAM(8-22)-spacer-H_(N)/A construct, thepCR 4 vector encoding the linker-BAM(8-22)-spacer insert is cleaved withBamHI+SalI restriction enzymes. This cleaved vector then serves as therecipient vector for insertion and ligation of the LC/A DNA (SEQ IDNO27) cleaved with BamHI+SalI. The resulting plasmid DNA is then cleavedwith PstI+XbaI restriction enzymes and serves as the recipient vectorfor the insertion and ligation of the H_(N)/A DNA (SEQ ID NO28) cleavedwith PstI+XbaI. The final construct contains theLC-linker-BAM(8-22)-spacer-H_(N)/A ORF for transfer into expressionvectors for expression to result in a fusion protein of the sequenceillustrated in SEQ ID NO125.

Preparation of Linker-Spacer-BAM8-22 Insert

The LC-H_(N) linker can be designed from first principle, using theexisting sequence information for the linker as the template. Forexample, the serotype A linker (in this case defined as the inter-domainpolypeptide region that exists between the cysteines of the disulphidebridge between LC and H_(N)) is 23 amino acids long and has the sequenceVRGIITSKTKSLDKGYNKALNDL. Within this sequence, it is understood thatproteolytic activation in nature leads to an H_(N) domain that has anN-terminus of the sequence ALNDL. This sequence information is freelyavailable from available database sources such as GenBank (accessionnumber P10845) or Swissprot (accession locus BXA1_CLOBO). Into thislinker an enterokinase site is incorporated; and using one of a varietyof reverse translation software tools [for example EditSeq best E. colireverse translation (DNASTAR Inc.), or Backtranslation tool v2.0(Entelechon)], the DNA sequence encoding the linker-ligand-spacer regionis determined. Restriction sites are then incorporated into the DNAsequence and can be arranged as BamHI-SalI-linker-proteasesite-PstI-XbaI-spacer-SpeI-BAM8-22-stop codon-HindIII. It is importantto ensure the correct reading frame is maintained for the spacer, BAMand restriction sequences and that the XbaI sequence is not preceded bythe bases, TC, which would result on DAM methylation. The DNA sequenceis screened for restriction sequence incorporation, and any additionalsequences are removed manually from the remaining sequence ensuringcommon E. coli codon usage is maintained. E. coli codon usage isassessed by reference to software programs such as Graphical Codon UsageAnalyser (Geneart), and the % GC content and codon usage ratio assessedby reference to published codon usage tables (for example, GenBankRelease 143, 13 Sep. 2004). This optimised DNA sequence is thencommercially synthesized (for example by Entelechon, Geneart orSigma-Genosys) and is provided in the pCR 4 vector.

Preparation of the LC/A-HN/A-BAM(8-22) Fusion

In order to create the LC/A-H_(N)/A-BAM(8-22) construct, the pCR 4vector encoding the linker-spacer-BAM(1-22) insert is cleaved withBamHI+SalI restriction enzymes. This cleaved vector then serves as therecipient vector for insertion and ligation of the LC/A DNA (SEQ IDNO27) cleaved with BamHI+SalI. The resulting plasmid DNA is then cleavedwith PstI+XbaI restriction enzymes and serves as the recipient vectorfor the insertion and ligation of the H_(N)/A DNA (SEQ ID NO28) cleavedwith PstI+XbaI. The final construct contains the LC/A-linker-proteasesite-H_(N)/A-spacer-SpeI-BAMS-22 ORF for transfer into expressionvectors for expression to result in a fusion protein of the sequenceillustrated in SEQ ID NO124.

Purification Method for BAM Fusion Proteins

The fusion proteins were purified as described in Example 54.

Example 59 Construction of CHO-K1 OPRM1 Binding Assay and NCI-H69SNAP-25 Cleavage Assay

A radioligand binding assay for measurement of the binding affinity(pK_(I)) of a range of reference ligands and fusions to human β-opioid(OP₃) receptors in CHO cells stably transfected with the human β-opioidreceptors (CHO-MOR cells) was used. The assay involves the specificlabelling of μ-opioid receptors (MOR) expressed in CHO cells with theMOR selective agonist radioligand [³H]-DAMGO. Increasing amounts ofunlabelled competing ligand (Syntaxin ligands or reference compounds)are incubated with a fixed amount of radioligand and cell membranes.Upon completion of the assay, reaction mixtures are filtered throughglass fibre paper where radioactivity associated with MOR receptorsbecomes trapped. The radioactivity of each filter is quantified and thisrelates to the ability of the competing ligand to bind to the MORreceptor in place of the radioligand. Ligands better able to bind to theMOR receptor will appear to ‘displace’ the radiolabel at lowerconcentrations.

Cell Culture

CHO-MOR cells (CHO-K1 cells with stable expression of the μ (OP3) PerkinElmer ES-542-C) were cultured according to the supplier'srecommendations. Cells were grown in Hams F12 containing 10% FBS and 400μg/ml G418 at 37° C. in an atmosphere of 95% O₂/5% CO₂. Cells werepassaged when cells had reached 80% confluency, typically every 3-4days.

Cells were grown in eight T175 flasks to ˜80% confluence, the culturemedium was removed and the cells washed twice with 10-20 ml PBS. Cellswere removed from the flask by addition of 5 ml of PBS based enzyme freedissociation buffer, and incubated at 37° C. for 10 min followed bygentle tapping of the flasks to dislodged cells. Ten ml of culture mediawas added to the flasks and the cells were transferred to a 50 ml Falcontube. Flasks were washed with a further 20 ml culture media which wasthen added to the cell suspension. Cells were centrifuged (1300×g, 3min), the supernatant removed, and the cell pellet resuspended inculture media by trituration. A cell sample was removed and the viablecell number calculated using a nucleocounter (ChemoMetec). The volume ofmedia was adjusted to give a final concentration of 5×10⁶ cells/ml. Oneml aliquots of the resultant cell suspension were transferred to 1.5 mlmicrofuge tubes and centrifuged (100×g, 5 min), the supernatant wasremoved and the cell pellets frozen and stored at −80° C.

Membrane Preparation

On the day of the assay, the required number of cell pellets was removedfrom −80° C. and 1 ml ice cold membrane preparation buffer (50 mM TrispH 7.4 at 4° C.) was added to each pellet. Individual cell pellets weredislodged by vortexing, collected into a 40 ml centrifuge tube and thetotal volumes adjusted to 10 ml. Cells were homogenised (1×1 s) using anUltra Turrax T25-digital homogeniser (IKA-WERKE), at the highest setting(25,000 min⁻¹). Membranes were diluted to the required cellconcentrations in Assay Buffer (50 mM Tris pH 6.96 at 21° C.) bycounting the total cells in a Nucleocounter and make to 300,000cells/ml.

Competition Binding Assay

In order to determine the binding profile of a number of β-opioidreceptor ligands or fusions, competition binding assays were performedin which a fixed concentration of [³H]-DAMGO was competed withincreasing concentrations of the β-opioid receptor ligands or fusionprotein. CHO-MOR cell membranes (equivalent to 30,000 cells per well)were incubated with a fixed concentration of [³H]-DAMGO (1 nM) andincreasing concentrations of competing ligand or fusion (0.01 nM to 1μM), in Assay Buffer (50 mM Tris pH 6.96 at 21° C.). To define thenon-specific and total binding, each reaction was performed in thepresence or absence of CTOP (1 μM). Reactions were performed, intriplicate, in deep-well ‘LoBind Protein’ 96-well plates (200 μl finalvolume) and initiated by addition of cell membranes. Assay plates weremixed, covered with a plate sealer and incubated for 1 hr at roomtemperature. Reactions were terminated by rapid filtration throughWhatman GF/B filters using a Brandel cell harvester, filters were washed(3×3 ml) with ice-cold wash buffer (50 mM Tris-HCl, pH 7.4 at 4° C.) andtransferred to scintillation vials. Scintillation fluid was added toeach vial and after 3 hours bound radioactivity was quantified in aTri-Carb 2900TR liquid scintillation analyser by counting each vial for3 min.

NCI-H69 SNAP-25 Cleavage Assay

SNARE cleavage by betaendorphin fusions was demonstrated by developing aNCI-H69 SNAP-25 cleavage assay. This assay used the human small celllung carcinoma cell line NCI-H69.

H69 cells were plated into poly-D-Lysine coated 96 well plates at 4×10⁵cells/ml. The plates were left for 24-48 hours prior to treatment. H69feeding Medium consisted of RPMI-1640 containing 10% FBS, 4.5 g/lGlucose, 1.5 g/l Sodium bicarbonate, 1 mM Sodium Pyruvate, 10 mM HEPES,2 mM Glutamine. 50 ml of cell media was filtered sterilised into asterile 50 ml centrifuge tube using a syringe and 0.2 um filter to beused to create two separate dilutions series for each TSI, with startingpoints at half log intervals (starting at 1 uM and 300 nM). The tworesulting series were then combined into one dose curve. Eachconcentration was plated in triplicate across the plate with a dosecurve running down the plate. By removing 125 ul media from the cellplate to be treated and adding 125 ul of each TSI solution to the well.Then the plate was placed in an incubator at 37° C., 5% CO₂ for 24hours. After 24 hours, test materials/feeding medium was removed fromthe plate by inverting over a waste receptacle and the remaining mediafrom each well was removed using a fine-tipped pastette. Next, the cellswere lysed using Lysis buffer (25% 4× NuPAGE LDS sample buffer, 10% 1MDTT, 65% dH2O); 100 μl lysis buffer was added to each well, and theplate left at room temperature for 5 minutes. Then the lysate wastransferred from each well into a 1.5 mL microcentrifuge tube using aGilson P200 pipette and placed in a heat block pre-warmed to 90° C. for10 minutes. SNAP-25 cleavage was then determined by western blottingwith a SNAP-25 antibody. 15 μL of lysed samples and 3 μL InvitrogenMagic Marker XP (LC5602) & 3 μL Invitrogen See Blue Plus 2 pre-stainedstandard (LC5925) was loaded onto Invitrogen 12% bis-tris 1 mm, 15 wellgels that were immersed in Invitrogen NuPAGE MOPS SDS Running Buffer.The gel was then run at 200 V until the Lysozyme 14 kDa marker is justabove the gel base (approximately 70 minutes). Transferred proteins fromthe gel to a nitrocellulose membrane on an iBlot dry blotting systemfrom invitrogen (IB1001UK), on program 2 (23 volts) for 6 minutes,according to the manufacturers instructions. On completion of the iBlotprogram, the membrane was to removed from the transfer stack and placedin a small tray containing blocking buffer (5 g Marvel milk powder per100 mL PBS/Tween 0.1%). The membrane was then incubated with blockingbuffer solution at room temperature, on a rocker, for 60 minutes. Afterblocking, the primary antibody solution was added to the blocking bufferand membrane; 10μ Anti-SNAP-25 (Sigma S-9684) added per 10 mL blockingbuffer (1:1000 dilution). Sigma's anti-SNAP-25 is reactive toward thewhole SNAP-25 protein so it therefore recognizes the intact and cleavedSNAP-25. Membranes were incubated with primary antibody at roomtemperature, on a rocker, for 60 minutes. Then the membranes were washedby performing 3 rinses with PBS/Tween 0.1% and further blocking bufferadded before incubating the membranes at room temperature, on a rocker,for 10 minutes. After incubation in blocking buffer the secondaryantibody was added to the membrane; 20 μl of Anti-Rabbit-HRP conjugate(Sigma A-6154) was added per 40 mL blocking buffer (1:2000 dilution).The membrane was incubate with secondary antibody at room temperature,on a rocker, for 60 minutes before being washed three times withPBS/Tween (0.1%). Again, further blocking buffer was added to themembrane and the membrane incubated at room temperature, on a rocker,for 30 minutes before being washed 3 times with PBS/Tween (0.1%).Finally, detection of bound antibody solution done using PierceWest-Dura supersignal (34075) detection reagents. The detection reagentswere mixed (Luminol/Enhancer Solution, Stable Peroxide Buffer) at a 1:1ratio (a total volume of 2 ml per mini membrane) and applied to themembrane, ensuring that the membrane is completely flat and the reagentscover it completely. The membrane was incubated for 5 minutes at roomtemperature before Chemiluminescent detection was performed on theGeneGnome HR Syngene system from Synoptics. The exposure was set to 5minutes and Gene tools software from Syngene was used to calculate therelative amounts of cleaved and uncleaved SNAP-25 within each lane.

Example 60 Construction of CHO-K1 BDKRB₁ and CHO-K1 BDKRB₂ ReceptorActivation Assay CHO-K1 BDKB₂ Receptor Activation Assay

A receptor activation assay was developed for which stably transfectedCHO-K1 cells with the B₂ receptor were used in a calcium fluorimetryassay measuring intracellular calcium levels. The assay allowed themeasurement of the potency (pEC₅₀) and intrinsic efficacy (E_(max)) ofthe bradykinin ligand and fusions. The assay involves indirectmeasurement of B₂-receptor activation by measuring changes inintracellular calcium levels using a Flexstation3 and calcium-sensitivedye.

Culture of CHO-K1 B2 Cells

CHO-K1 cells with stable expression of the B₂ receptor (CHO-K1-B₂-R;ES-090-C) were purchased from Perkin Elmer. Cells were cultured in Ham'sF12 containing 2 mM glutamine, 10% FBS and 400 μg/ml G418 at 37° C. in ahumidified environment containing 5% CO₂. Cells were passaged every 3 to5 days when cells were ˜80% confluent. The media was removed and thecells washed twice with PBS. Cells were harvested using a PBS-basednon-enzymatic cell dissociation buffer at 37° C. for 2-3 minutes,pelleted by centrifugation, resuspended in culture media and seeded intofresh T175 flasks.

Seeding of CHO-K1 B2 Cells

Cells were harvested using a PBS-based non-enzymatic cell dissociationbuffer at 37° C. for 2-3 minutes. Cells were collected bycentrifugation, resuspended in culture media and the cell concentrationdetermined using a nucleocounter (ChemoMetec). Cells were diluted inculture media to the required concentration of 2×105 cells ml⁻¹ andseeded into 96-well plates at a volume of 100 μl per well. Cells wereincubated at 37° C. in 5% CO₂ overnight.

Estimation of Potency and Intrinsic Activity of Bradykinin and BKFusions

The following day after seeding, culture media was removed from thecells and replaced with 100 μl per well of assay buffer (HBSS with 1.26mM CaCl₂, 0.49 mM MgCl₂, 0.4 mM MgSO₄ and 20 mM HEPES at pH 7.4)containing 5 mM probenecid (probenecid final concentration of 2.5 mM)and FLIPR calcium 4 loading dye (100 μl). Cells were incubated at 37° C.in 5% CO₂ for 60 min after which increasing concentrations of bradykinin(50 μl) or fusion protein were added to the cells in triplicate by theFlexstation3. The change in fluorescent emission at 525 nm followingexcitation at 485 nm was determined over a 70 s time period using theFlexStation3.

CHO-K1 BDKB₁ Receptor Activation Assay

An assay was also developed to allow the measurement of potency (pEC₅₀)and efficacy (Emax) of bradykinin ligands at the human bradykinin B₁receptor stably expressed in CHO-K1 cells. This assay was similar to theB₂ receptor activation assay as it measured is the changes inintracellular calcium levels using a calcium fluorimetry assay. CHO-K1cells with stable expression of the B₁ receptor were purchased fromPerkin Elmer (ES-091-C).

Culture of CHO-K1 B1 Cells

CHO-K1 cells with stable expression of the human B1 receptor (CHO-K1-B1cells) were cultured in culture media (Ham's F12 containing 2 mMglutamine, 10% FBS and 400 μg·ml−1 G418) at 37° C. in a humidifiedenvironment containing 5% CO₂. Cells were passaged every 3 to 5 dayswhen cells were ˜80% confluent. The media was removed and the cellswashed twice with PBS. Cells were harvested using a PBS-basednon-enzymatic cell dissociation buffer at 37° C. for 2-3 min, pelletedby centrifugation (1,500 rpm; 3 min), re-suspended in culture media andseeded into fresh T500 flasks. Pellet cells by centrifugation at 1,300rpm for 3 min in a Hereaus megafuge 1.0. Then the cell pellet wasresuspended in Bambanker freezing medium to achieve a concentration of3×10⁶ cells·mL⁻¹ and aliquoted into 1.8 mL Nunc cryovials. The cryovialswere transferred to a cryo vial rack and stored at −80° C. overnightbefore transferring the vials to the short-term liquid nitrogen store.

Seeding of CHO-K1 B1 Cells

The day before the assay, cell vials were removed from the liquidnitrogen store and thawed quickly by placing vials in a 37° C. waterbath. Cells were then pelleted by centrifuging at 1,300 rpm for 3 min ina Hereaus megafuge 1.0. The cell pellet was re-suspend in 2 mLmedia/vial and the number of viable cells in the suspension countedusing the Nucleocounter. Added media to the cell suspension to achieve acell concentration of 1×10⁶ cells·mL⁻1. Then the cells were plated in aCorning black walled, clear-bottom, half-area plate at a density of20,000 viable cells·well⁻¹; using a Rainin E8-300 multi-channel pipetteadded 30 μL media to each well followed by 20 μL·well⁻¹ of the 1×10⁶cells·mL⁻¹ cell suspension. Maintained the plates overnight at 37° C. ina humidified environment containing 5% CO₂.

Estimation of Potency and Intrinsic Activity of des-Arg Bradykinin andBK Fusions

Next day the plates were incubated (37° C. in a humidified environmentcontaining 5% CO₂) in HBSS modified assay buffer (with 1.26 mM CaCl₂,0.49 mM MgCl₂, 0.4 mM MgSO₄ and 20 mM HEPES at pH 7.4) containing x0.5Ca²⁺-dye, probenecid (2.5 mM). After 1 hour, increasing concentrationsof des-Arg⁹-BK and fusion proteins were added to the cells in triplicaterows by the FlexStation3® (height 70 μl; speed 16 μl·s⁻¹; 37° C.). Thefluorescence emitted at 525 nm was measured over a 60 s time period andexpressed as % increase in baseline RFU.

Example 61 Construction and Activation of Bradykinin Fusion Proteins

Following the methods used in Examples 18 and 19, the LC/A (SEQ ID NO27)and H_(N)/A (SEQ ID NO28) were created and inserted into the A serotypebradykinin linker arranged as BamHI-SalI-linker-proteasesite-PstI-XbaI-spacer-SpeI-bradykinin-stop codon-HindIII. The finalconstruct contained the LC/A-linker-proteasesite-HN/A-spacer-SpeI-Bradykinin ORF (SEQ ID NO 131) for expression as aprotein of the sequence illustrated in SEQ ID NO132.

Alternatively, the bradykinin (SEQ ID NO129) in the bradykinin linkerwas replaced by des Arg⁹-bradykinin (SEQ ID NO 130) so that the finalconstruct contained the LC/A-linker-protease site-HN/A-spacer-SpeI-desArg⁹-Bradykinin ORF for expression as a protein of the sequenceillustrated in SEQ ID NO133.

Purification Method for Bradykinin Fusion Proteins

The fusion proteins were purified as described in Example 54.

Example 62 Construction and Activation of Substance P Fusion Proteins

Following the methods used in Examples 18 and 19, the LC/A (SEQ ID NO27)and H_(N)/A (SEQ ID NO28) are created and inserted into the A serotypesubstance P analogue (S6) linker arranged as BamHI-Sail-linker-proteasesite-PstI-XbaI-spacer-SpeI-substance P (S6)-stop codon-HindIII. Thefinal construct contains the LC/A-linker-proteasesite-HN/A-spacer-SpeI-substance P(S6) ORF for expression as a protein ofthe sequence illustrated in SEQ ID NO136.

Purification Method for Substance P Fusion Proteins

The fusion proteins were purified as described in Example 54.

Example 63

A method of treating, preventing or ameliorating pain in a subject,comprising administration to said patient a therapeutic effective amountof non-cytotoxic protein conjugate, wherein said pain is selected fromthe group consisting of: chronic pain arising from malignant disease,chronic pain not caused by malignant disease (peripheral neuropathies).

Patient A

A 73 year old woman suffering from severe pain caused by posthepaticneuralgia is treated by a peripheral injection with non-cytotoxicprotein conjugate to reduce neurotransmitter release at the synapse ofnerve terminals to reduce the pain. The patient experiences goodanalgesic effect within 2 hours of said injection.

Patient B

A 32 year old male suffering from phantom limb pain after having hisleft arm amputated following a car accident is treated by peripheralinjection with non-cytotoxic protein conjugate to reduce the pain. Thepatient experiences good analgesic effect within 1 hour of saidinjection.

Patient C

A 55 year male suffering from diabetic neuropathy is treated by aperipheral injection with non-cytotoxic protein conjugate to reduceneurotransmitter release at the synapse of nerve terminals to reduce thepain. The patient experiences good analgesic effect within 4 hours ofsaid injection.

Patient D

A 63 year old woman suffering from cancer pain is treated by aperipheral injection with non-cytotoxic protein conjugate to reduceneurotransmitter release at the synapse of nerve terminals to reduce thepain. The patient experiences good analgesic effect within 4 hours ofsaid injection.

All documents, books, manuals, papers, patents, published patentapplications, guides, abstracts and other reference materials citedherein are incorporated by reference in their entirety. While theforegoing specification teaches the principles of the present invention,with examples provided for the purpose of illustration, it will beappreciated by one skilled in the art from reading this disclosure thatvarious changes in form and detail can be made without departing fromthe true scope of the invention.

1. A non-cytotoxic protein conjugate for inhibition or reduction ofexocytic fusion in a nociceptive sensory afferent cell, comprising: (i)a Targeting Moiety (TM), wherein said TM is capable of binding to aBinding site on a nociceptive sensory afferent cell, wherein saidBinding site undergoes endocytosis to be incorporated into an endosomewithin the nociceptive sensory afferent cell, and wherein the TM isselected from the group consisting of BAM, β-endorphin, bradykinin,substance P, dynorphin and/or nociceptin; (ii) a non-cytotoxic proteaseor a fragment thereof, wherein the protease or protease fragment cleavesa protein of the exocytic fusion apparatus of said nociceptive sensoryafferent cell; and (iii) a Translocation Domain, wherein theTranslocation Domain translocates the protease or protease fragment fromwithin the endosome, across the endosomal membrane, and into the cytosolof the nociceptive sensory afferent cell.
 2. The non-cytotoxic proteinconjugate of claim 1, wherein the non-cytotoxic protease is selectedfrom a clostridial neurotoxin, or an IgA protease.
 3. The non-cytotoxicprotein conjugate of claim 1, wherein the Translocation Domain is abotulinum H_(N) domain.
 4. The non-cytotoxic protein conjugate of claim1, wherein the TM binds specifically to one or more receptors selectedfrom the group consisting of Mrg receptors such as MrgX1, opiodreceptors such as OPRD1 and/or OPRM1, BDKRB1 and/or BDKRB2, Tachykininreceptors such as TACR1, TACR2 and/or TACR3, Kappa receptor (OPRK1) andORL₁ receptor.
 5. The non-cytotoxic protein conjugate of claim 1,wherein the TM comprises an amino acid sequence having at least 70%sequence identity to one or more amino acid sequences selected from thegroup consisting of SEQ ID NOs: 2, 4, 12, 101, 120, 121, 126, 129, 130,134 and
 135. 6. The non-cytotoxic protein conjugate of claim 1, whereinthe nociceptive sensory afferent cell is a primary nociceptive sensoryafferent cell.
 7. The non-cytotoxic protein conjugate of claim 1,wherein said conjugate comprises an amino acid sequence having at least90% sequence identity to one or more amino acid sequences selected fromthe group consisting of SEQ ID NOs: 50, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 123, 124, 125, 127, 128, 132, 133, 136, 137, 138,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,and
 169. 8. A pharmaceutical composition comprising a non-cytotoxicprotein conjugate according to claim 1 and a pharmaceutically acceptablecarrier.
 9. A polynucleotide molecule encoding the non-cytotoxic proteinconjugate of claim
 1. 10. A method of preparing a non-cytotoxic proteinconjugate comprising expressing the polynucleotide of claim 9 in a hostcell.
 11. A method for treating, preventing or ameliorating pain in asubject, comprising administering to said subject a therapeuticallyeffective amount of a non-cytotoxic protein conjugate according toclaim
 1. 12. The method according to claim 11, wherein the pain ischronic pain selected from the group consisting of neuropathic pain,inflammatory pain, headache pain, somatic pain, visceral pain andreferred pain.
 13. A method for treating, preventing or amelioratingpain in a subject, comprising administering to said subject atherapeutically effective amount of a pharmaceutical compositionaccording to claim
 8. 14. The method according to claim 13, wherein thepain is chronic pain selected from the group consisting of neuropathicpain, inflammatory pain, headache pain, somatic pain, visceral pain andreferred pain.